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PHYSIOLOCIICAL CHEMISTRY
AN INTERMEDIATE TEXTBOOK OF
PHYSIOLOGICAL CHEMISTRY
WITH EXPERIMENTS
BY
C. J. V. P
JPJ]TTIBONE, Ph.D.
ASSOCIATE PROFESSOR OF PHYSIOLOGICAL CHEMISTRY, MEDICAL SCHOOL, UNIVERSITY OF MINNESOTA, MINNEAPOLIS
SECOND EDITION
ST. LOUIS
C. V. MOSBY COMPANY
1922
Copyright, 1917, 1922, Bv C. V. Mosbv Company (Printed in U. S. A.)
Press of
.C. V. Mosby Company,
St. Louis.
ri4
TO
M. A. P., I. P. H., AND M. P. G.
:l?7l+*
PREFACE TO SECOND EDITION
In preparing the second edition of this book the author lias attempted to preserve the original character of the work. Much material has been added, however, both in the text and labora- tory section, and some older methods have been omitted. A chapter on the portions of physical chemistry of most interest in biology has been added, including more extended discussion of colloids, and the material on enzymes has been considerably extended. More material on the saliva, and muscle has been included and a summary of the vitamines to the present date. In the laboratory section the principal additions are the segre- gation of the physicochemical material with the addition of vari- ous simple but significant experiments, and the inclusion of Folin 's methods for blood analysis and microchemical estimation of certain constituents of the urine.
The author wishes to express thanks for various suggestions and corrections.
C. J. V. P.
Minneapolis, August, 1922.
PREFACE TO FIRST EDITION
My aim in writing this book has been to prepare an inter- mediate text which would cover the general field of physiologi- cal chemistry in such a way as to give students a familiarity with compounds important from a biochemical viewpoint, and to acquaint them with the fundamental processes which go on in the animal body. I have attempted to avoid confusing the beginner with lengthy discussions of debated points, but to set forth as clearly as possible the present status of our knowledge. The material is so chosen that the book may be used for inter- mediate classes, or for advanced work if supplemented by lec- tures.
9
10 PREFACE
The appended laboratory work has been drawn from the manual in use in my classes for the last five years. Much of the material has, of course, been drawn from other manuals.
I wish to acknowledge suggestions and corrections in the laboratory directions from various colleagues, particularly Dr. F. B. Kingsbury. I wish also to express my thanks to Dr. W. H. Hunter, who kindly volunteered to read the manuscript of the theoretical portion.
C. J. V. Pettibone.
Minneapolis, Minn.
CONTENTS
PART I
INTBODUCTOEY
Object and Importance of Physiological Chemistry, 17; Protoplasm, 18; Material Bases, 19.
CHAPTER I
Physical Chemistry in Its Relations to Physiological Chemistry
Importance, 21; Osmotic Pressure, 21; Electrical Properties of Solu- tions, 23; H-ion Concentration, 23; Titratable Acidity, 25; Colloidal Solu- tions, 28; Classification and Properties of Colloids, 29; Tyndall's Phe- nomenon, 30 ; Electrical Properties of Colloids, 31 ; Methods of Precipitating Colloids, 32; Absorption — Surface Tension, 33; Imbibition, 34.
CHAPTER II
Elements, Inorganic Materials, Water
Elements Found in the Body, 35; Importance not Determined by Amount Present, 35; Carbon, Hydrogen, Oxygen, Nitrogen, Sulphur and Phosphorus, 36; Sodium, Potassium, Calcium, Magnesium and Iron, 38; Chlorine, Iodine, Fluorine, etc., 40; Water, 40.
CHAPTER III
Carbohydrates
Composition, Occurrence, General Function, 41 ; Structure of the Car- bohydrates, 41; Optical Activity, 42; Classification of Carbohydrates, 48; Origin and Synthesis, 49; Interconversion of Carbohydrates, 51; Combina- tion of Carbohydrates with One Another, and with Other Substances, 52; Behavior with Strong Alkalies, 52; Behavior with Acids, 53; Oxidation of Carbohydrates, 54; Reduction of Carbohydrates, 57; Formation of Osa- zones, 57; The Molisch Test, 58; Fermentation — Enzymes, 59; Nomencla- ture and Classification, 62 ; Specific Nature, 63 ; Influence of Temperature, 64; Effect of Chemical Reaction, 64; Reversibility, 65; Active and Inactive
11
12 CONTENTS
Form, 65; Action Retarded by Products, 66; Progressive Action, 66; Anti- enzymes — Defensive Enzymes, 66; Summary, 66; Individual Groups of Carbohydrates, 67 ; Pentoses, 67 ; Absorption Spectra, 68 ; Hexoses. CgH^jOg, 69; Glucose, 69; Fructose. (Levulose, Fruit Sugar), 70; d-Galactose, 7(>; Amino Sugars, 71; d-Glycuronic Acid, 71; Disaccharides, 72; Saccharose. (Siucrose, Cane Sugar), 72; Lactose, 74; Maltose, 75; Polysaccharides, 75; Starch (CJI^J^^)^, 76; Dextrins, 77; Inulin, 77; Gums and Mucilages, 77; Cellulose, 77; Glycogen, 78; Glucosides, 79.
CHAPTER IV
Fats, Phosphatids, and Allied Substances
Distribution and Importance, 80; Composition and Structure, 80; General Properties, 82 ; Emulsification, 82 ; Saponification, 83 ; Rancid Fats, 84; Detection and Identification, 84; Important Fats, 86; Phosphatids, Cerebrosides, and Sterols, 87; Lecithin, 88; Cholesterol, 89.
CHAPTER V
Proteins
Introductory, 91 ; Elementary Composition, 91 ; Classification, 92 ; Preparation of Proteins from Materials in Which Tliey Occur, 93; Molecu- lar Weight, 94; Hydrolysis, 94; Amino Acids Obtained by Hydrolyzing Protein, 95; General Properties and Reactions of Amino Acids, 98; Gen- eral Protein Reactions, 101 ; Color Tests, 101 ; Precipitation Reactions, 103 ; Structure of the Protein Molecule, 105; Putrefaction of Proteins, 109; Individual Groups, Simple Proteins, 110; Albumins, 111; Globulins, 111; Glutelins, 112; Prolamines, 112; Albuminoids, 112; Histones, 113; Prota- mines, 113; Conjugated Proteins, 113; Glycoproteins, 114; Phosphoproteins, 114; Hemoglobins, 116; Detection of Hemoglobin, 118; Absorption S'pectra of Oxyhemoglobin and Hemoglobin, 120; Derivatives of the Hemoglobins, 121; Fate of Blood Pigment in the Body, 123; Nucleoproteins, 123; Lecithoproteins, 125; Derived Proteins, 125; Primary Protein Derivatives, 125; Secondary Protein Derivatives, 127.
CHAPTER VI
Some Familiar Foodstuffs — Some Important Tissues
Some Important Foodstuffs, 130; Cooking and Preparation of Foods, 131; Milk, 131; Butter, 132; Clieese, 133; Meats, 133; Eggs, 133; Vege- tables, 134; Breadstuffs, 134; Choice of Diet, 135; Some Important Tis- sues, 135; Muscle, 135; Brain and Nerves, 138; Bones and Teeth, 138; Connective Tissue, 139; The Blood, 140; Reaction of the Blood, 142; Os-
CONTENTS 13
motic Pressure, 143; Coagulation of tlie B-lood, 143; Lymph, 144; The Skin, 144.
CHAPTER VII
Digestion in the Mouth General Purpose of Digestion, 145; Preparation of Food, 146; Saliva, 146.
CHAPTER VIII
Digestion in the Stomach Importance, 152 ; Methods of Study, 152 ; Causation of Flow of Gastric Juice, 153; General Character of the Secretion, 155; Hydrochloric Acid, 155; Source of the Hydrochloric Acid, 157; The Functions of the Hydro- chloric Acid, 157; Enzymes of the Gastric Juice, 158; Pepsin, 158; Prod- ucts of Peptic Digestion, 159; Rennin, 159; Are Pepsin and Rennin Iden- tical? 160; The Stomach Wall is not Digested, 161; Passage of the Food into the Intestine, 161.
CHAPTER IX Digestion in the Intestine General, 162; Pancreatic Juice, 162; General, 162; Mechanism of Flow, 163; Composition of Pancreatic Juice, 163; Tiypsin, 164; Rennin, 165; Action on Fats, 165; Steapsin, 165; Action on Starches, 165; Amylase or *'Amylopsin," 165; The Bile, 166; Causes of Flow, 166; Amount, 166; Composition. Function, 167; Bile Pigments, 167; Bile Salts, 168; Intesh tinal Secretion, 168; Erepsin, 169; Other Enzymes, 169; Excretory Func- tion of Intestinal Secretion, 169; Bacterial Action in the Intestine, 17Q; Feces, 171.
CHAPTER X
Absorption General, 173; Absorption of Proteins, 174; Carbohydrate Absorption, 174; Absorption of Fats, 175.
CHAPTER XI
Urine General, 176; Physical Properties, 177; Volume, 177; Oolior, Trans- parency, 178; Consistency, Odor, Taste, 179; Specific Gravity. — Total Solids, 179; Optical Activity, Reducing Power, Fermentation, etc., 180; Reaction, 181; Urea, 182; Uric Acid and Other Purine Derivatives, 184; Hippuric Acid, 187; Ammonia, 188; Creatinine and Creatine, 188; Inor- ganic Constituents, 190; Chlorides, 190; Phosphates, 191; Sulphates, 191;
14 CONTENTS
Carbonates, 193; Sodium, Potassium, Calcium and Magnesium, 193; Patho- logical Constituents of the Urine, 193.
CHAPTER XII
Metabolism
Oeneral, 194; Protein Metabolism, 195; Amount of Protein Required. Nitrogen Balance, 197; Carbohydrate Metabolism, 203; Sources of Gly- cogen, 209; Metabolism of Fats, 211; Metabolism of Inorganic Material, 213; Energy Exchange, 214; Utilization of Alcohol by the Body, 220; Starvation, 220; Unknown Food Constituents. — Vitamins, 221; Body Tem- perature, 225; Inlluence of Organs of Internal Secretion, 225.
PART II LABORATORY WORK
Mnterials, 228; General Laboratory Instructions, 234.
CHAPTER I Standard Acid and Alkali Physical Chemistry — Calibration of Volumetric Apparatus, 236; Nor- mal Acid and Alkali, 236; Oxalic Acid Method, 237; Sodium Carbonate Metliod, 238; Preparation of N/10 Acid, 239; Preparation of a Standard Alkali, 240; Kjeldahl Method for Nitrogen Determination, 241; Indicators, 244 ; Catalysis, 244 ; Colloids, 245 ; Solation and Gelation, 245 ; Imbibition, 245; Dialysis, 245; Suspensoids and Emulsoids, 245; Reversibility, 245; Diffusion, 245; Absorption, 246.
CHAPTER II
Detection of the Elements and of Inorganic Salts Carbon, 247; Hydrogen, 247; Oxygen, 247; Nitrogen, 248; Sulphur, 248'; Preparation of Muscle Extract, 248; Muscle Residue, 249; Biood Serum, 249; Bone, 250; Tests for Inorganic Materials, 250; Chlorides, 250; Sulphates, 251; Phospliates, 251; Carbonates, 251; Calcium, 251; Mag- nesium, 251; Iron, 252; Sodium, 252; Potassium, 253.
CHAPTER III Carbohydrates
Monosaccharides, 254; Dextrose, 254; Benedict's Qualitative Reagent for Sugar, 255; Levulose, 258; Galactose, 259; Arabinose, 259; Disac-
CONTENTS 15
cliarides, 260; Saccharose, (Sucrose), 260; Maltose, 260; Lactose, 261; Polysaccharides, 261; Starches, 261; Dextrines, 262; Glycogen, 263.
CHAPTER IV
Fats and Phosphatides Fats, 264; Phosphatides, 267; Lecithin, 267.
CHAPTER V
Proteins
General Protein Reactions, 269; Color Reactions, 269; Precipitation Tests, 271; Individual Groups — Simple Proteins, 274; Albumins, 274; Globulins, 275; Prolamines, 276; Gluteins, 277; Albuminoids, 277; His- tones, 278; Protamines, 278; Conjugated Proteins, 279; Glycoproteins, 279; Hemoglobins, 279; Phosphoproteins, 286; Nucleoproteins, 288; De- rived Proteins, 289; Primary Protein Derivatives, 289; Proteans, 289; Metaproteins, 289; Coagulated Proteins, 291; Secondary Protein Deriva- tives, 291; Proteoses, 291; Peptones, 292; Peptids, 293; Amino Acids, 203.
CHAPTER VI
MiCROCHEMICAL METHODS FOR B'LOOD ANALYSIS
Folin's Modified Nessler's Reagent, 296; Preparation of Protein- Free Blood Filtrates, 297; Nonprotein Nitrogen Determination, 299; Determina- tion of Urea of Urease Decomposition and Distillation, 300; Determination of Preformed Creatinine, 302; Determination of Creatine Plus Creatinine, 303; Determination of Uric Acid in Blood, 304; Blood Sugar, 306; Method for the Determination of Chlorides in Blood Plasma, 309.
CHAPTER VII
Salivary Digestion Composition, 310; Digestive Action, 311.
CHAPTER VIII
Gastric Digestion
Preparation of Artificial Gastric Juice, 314; Composition of Gastric Juice, 314; Digestive Action of Gastric Juice, 316; Motor Power of the Stomach, 318; Rate of Absorption from the Stomach, 318,
16 CONTENTS
CHAPTER IX
PANCRKA.TIO DIGESTION — BiLE
Pancreatic Juice, 319; Cbmposition of Pancreatic Juice, 319; Diges- tive Action, 319; Intestinal Juice, 320; Bile, 320; Composition, 320; Ef- fect of Bile on Surface Tension, 321; Biliary Calculi, or Gall Stones, 321.
CHAPTER X Urine
Qualitative Study, 323; Inorganic Constituents, 323; Organic Con- stituents, 325; Collection and Preservation of a Specimen for Quantitative Analysis — General Properties, 328; Quantitative Analysis (Make All De- terminations in Duplicate), 331; Total Solids, 331; Total Nitrogen, 332; Ammonia, 332; Urea, 334; Colorimetric Method for Determination of Urea in Urine, 335; Uric Acid, 337; Hippuric Acid, 340; Purine Bases, 340; Creatinine (Folin), 340; Microchemieal Determination of Creatinine in Urine, 342; Indican, 343; Allantoin, 343; Oxalic Acid, 343; Chlorides, 343; Sulphates, 344; Phosphates, 345; Pathologic Urine, 346; Carbohy- drates, 349; Acetone Bodies, 353.
APPENDIX
Directions for Making Up Quantitative or Special Reagents Ammonium Thiocyanate, Standard, for Chlorides, 354; Barfoed's Solu- tion, 354; Barium Chloride for Sulphate Determination, 354; Benedict's Solution for Carbohydrate Estimation, 355; Congo Paper, 355; Esbach's Reagent, 355; Fehling's Solution (Quantitative), 355; Fehling's Solution (Qualitative), 355; Folin-Schaffer Reagent, 355; Formalin (Neutral), 356; Glyoxylic Acid Solution, 356; Guenzberg's Reagent, 356; Magnesia Mix- ture, 356; Mett's Tubes, 356; Millon's Reagent, 356; MoUsch's Re- agent, 357; Nylander's Reagent, 357; Pancreatic Solution, 357; Pepsin Solution, 357; Potassium Bichromate N/2, 357; Potassium Permanganate N/20, 357; Potassium Pyroantimonate K^RjSb^O,, 357; Silver Nitrate (Standard) for Volhard Chloride Method, 357; Sodium Cobaltinitrite NagCo (N02)e, 357; Special Sodium Acetate Solution (For Uranium Ace- tate Method for Phosphates), 358; Stokes' Reagent, 358; Toepfer's Re- agent, 358; Uranium Acetate Solution (Standard), 358.
PHYSIOLOGICAL CHEMISTRY
PART I
INTRODUCTORY
The scientific field known variously as Physiological, Biologi- cal or Biochemistry is the branch of science which treats of the chemical constitution, reactions and products of living ma- terial, whether of animal or plant origin. There is a growing tendency to use the terms Biological and Biochemistry to de- note the entire field, and to restrict the term Physiological chemistry to that portion of the subject dealing with animal material, but this practice is by no means general. It was once believed that the chemical processes going on in plants and ani- mals were fundamentally different. Synthesis or building up was considered characteristic of plants, whereas animals were known to desynthesize or break down the substances which they ate. We now know that this difference is a quantitative anc! not a qualitative one, for if kept in the dark, plants take up oxygen, burn their constituents and give off carbon dioxide in a manner analogous to the process predominating in animals. Animals, on the other hand, are now known to be capable of performing numerous and elaborate syntheses, breaking down the materials of their food to simpler compounds, but rebuild- ing many of the fragments into tissue substance, or altering them to produce compounds having specialized biological func- tions.
Object and Importance of Physiological Chemistry. — The ultimate object of workers in the field is to establish a rela-
17
18
PHYSIOLOGICAL CHEMISTRY
tionship between chemical composition and biological function, to be able to explain the workings of cells or of the various or- gans and tissues in terms of chemical reactions, but experi- mentors are still far from the attainment of this end, although many problems now are clearly understood which a few years ago were still unsolved. The findings of physiological chem- ists, and the methods of analysis developed by them have been of the greatest value to the science of medicine in general, and to the medical practitioner in particular. Since the body is made up of chemical compounds, and since many of its activ- ities depend upon chemical reactions, it is obvious that any light thrown upon the nature and properties of its components will tend to make clearer the character of the reactions in- volved in the normal functioning of the tissues, thus furnishing a basis for the study and correction of abnormal or diseased conditions. Both diagnosis and treatment have come to depend more and more upon the findings of the physiological chem- ists, and the general advancement of medicine has been greatly furthered by the results of biochemical research.
Protoplasm. — Living material, whether of plant or animal origin has been found to consist of a substance which is strik- ingly uniform throughout the entire living world. This ma- terial has been given the name protoplasm (from the Greek words meaning ^^ first," and ^^form"). It is a jelly-like watery mass, sometimes fairly rigid in form, possessing cer- tain characteristics which serve to distinguish living from life- less material. The first of these is the power of growth, — growth from internal forces such as we observe in animals and plants, and not growth from without such as the enlarging of a crystal. The second is the power of respiration, — taking up oxygen and giving ofP carbon dioxide. The third is the power of movement, — from place to place in animals, and movement incident to growth in plants. The fourth is irritability, and the fifth the power of reproduction. All living material possesses these five properties, and no lifeless material possesses all of them. Physiological chemistry may be looked upon as the
INTRODUCTION 19
study of the chemistry of protoplasm, its products and the sub- stances which it requires for the continuance of its normal functions.
Material Bases. — Amounts in Body. — In beginning the study of so broad and complicated a field it is difficult to choose a point of attack. The most satisfactory plan will be first to be- come familiar with the chemical substances out of which living material is made up. The number of these compounds is nat- urally large, but for convenience they may be classified in five great groups which are given the name of the Base Materials, or Material Bases.
The five groups are as follows:
I. Inorganic materials including water.
II. Carbohydrates.
III. Fats, Phosphatids and related compounds.
IV. Proteins.
v. Extractives.
Our first task will be to become familiar with the character- istics and properties of the Material Bases, studying group reactions and also the specific properties of important individ- ual compounds, and methods for their detection and estima- tion. We will then trace the history of the various substances in their passage through the body, considering their fate in the alimentary canal, their subsequent behavior as constitu- ents in the body tissues and fluids, and the final elimination of end products formed by their destruction.
The relative amounts of the different classes of Material Bases in the animal body are somewhat variable. "Water and other inorganic materials make up about 65-70% of the entire body weight of which only 4.5-5% is ash and the remainder water. Carbohydrates are present only in small quantities, the amount being less than 1%. Fats and related compounds vary considerably in amount with the general state of the body, since they may be stored away in large quantities. The body contains on the average about 15%. Proteins vary less from
20
PHYSIOLOGICAL CHEMISTRY
an absolute standpoint, but relatively the percentage depends upon the amount of fat. The amount in the body averages also about 15%. Extractives, a heterogeneous group of com- pounds classed together because they are water soluble, and belong in none of the other classes, make up less than 1% of the body weight. This group will receive no further treat- ment as such, but the substances included in it (urea, creat- inine, etc.) will be discussed individually in connection with the tissues or fluids in which they occur.
CHAPTER I
PHYSICAL CHEMISTRY IN ITS RELATIONS TO PHYSIOLOGICAL CHEMISTRY
Importance. — Among the most striking advances in physio- logical chemistry in recent years are those in which the methods and principles of physical chemistry have been brought to the aid of biochemists. Investigation of the properties of solutions, of dissociation, osmotic pressure, surface tension, of colloidal solutions, adsorption, dift'nsion, mass action, ionic equilibrium, hydrogen-ion concentration and many other fields have direct bearing on problems connected with the functioning of the cells and tissues. In fact the continuance of cell activity is insep- arably bound up with physicochemical phenomena, — absorption, secretion, excretion, growth, muscular contraction and an end- less list of other functions of living matter are carried out in conformity with physicochemical laws. The importance of physical chemistry in biochemical work is thus apparent. It is not within the scope of this book to enter into extended dis- cussion of physicochemical fields. However, some of the more important phases of the subject will be presented briefly.
Osmotic Pressure. — It is a well-known fact that a gas exerts a definite pressure, and that this pressure is inversely propor- tional to the volume. If the volume is halved, the pressure is doubled, etc. The volume of a gas allowed to expand increases %73 of its volume at 0°C. for a rise of 1°C. Also if an amount of a gas equal in grams to the figure expressing its molecular weight is contained in a 22.4 liter container, it will exert 1 atmosphere pressure. These facts are expressed as the funda- mental gas laws.
It has been discovered that dissolved substances also exert a pressure which in many respects behaves in accordance with
21
22
PHYSIOLOGICAL CHEMISTRY
the gas laws. This pressure of a dissolved substance is called osmotic pressure. Obviously it will be impossible to measure this pressure by observing the pressure of the solution on its container. It will be necessary to devise a separating wall or membrane through which the solvent, but not the dissolved sub- stance can pass. Such a membrane is called a semipermeable membrane. A sheet of parchment or a thin film of collodion serves such a purpose. Since the solvent can pass through the membrane, it will tend to pass tlirough the membrane freely. If an apparatus is set up with such a membrane, on one side of which is a solution, on the other pure solvent, the solvent will pass into the solution faster than the reverse process, and tend to dilute the solution. It is possible to place the solution in connection with a manometer, and thus measure the osmotic pressure. Osmotic pressure is directly proportional to the amount of dissolved substance, and to the absolute temperature, at least for dilute solutions. Aside from its usefulness in deter- mining molecular weights and other theoretically important data, it is at once apparent that osmotic pressure will play an enormously important role in the body. The body and cell fluids are solutions, and the cell membranes are semipermeable membranes. The maintenance of a proper distribution of the liquids in the body depends on a delicate balance of osmotic equilibrium, as do also such processes as secretion, absorption, etc.
A biological method of measuring osmotic pressure consists in immersing cells in a liquid of known osmotic pressure. If the pressure in the surrounding liquid is higher than that in the ceU, water passes out of the cell, and its contents can be seen to shrink away from the cell wall. If the reverse is the case, water passes into the cell, even to the point of rupture. The passage of water throug'^h the membrane is called osmosis. The above described method of determining it is called plasmolysis.
If the pores of the membrane are large enough to allow the passage not only of water molecules, but also of simple chemical substances such as salts, but still too small to allow the passage
PHYSICAL CHEMISTRY 23
of larger molecules such as those of sugar, proteins, etc., the process is called dialysis. Dialysis is of value in freeing from salts, etc., a solution of protein or other nondialyzable sub- stance.
There has been much discussion as to whether osmosis and diffusion or these plus simple filtration could account for the absorption from the intestine, the excretion of urine, the forma- tion of lymph, the passage of gases through the alveolar walls in the lungs and many other processes. Whereas these phe- nomena doubtless are influenced by the laws governing osmosis and diffusion, they evidently are controlled also by other fac- tors. For example an isolated loop of intestine will absorb substances from within itself which are present in concentration no greater than that in which they exist in the blood, whereas the concentration of many substances in the urine is much above their concentration in the blood. On the other hand, the trans- ference of gases in the lungs probably follows simple laws of diffusion except in time of stress, when cellular activity appears to come into play.
Electrical Properties of Solutions. — It is a well established fact that substances in solution often carry electric charges. This can be demonstrated by passing an electrical current through a solution. The substances in solution will migrate toward the positive or negative pole according as they carry negative or positive charges. This migration in the current is called cataphoresis. Only substances which are dissociated into their ions will migrate thus. Those which are not dissociated do not migrate, and thus do not conduct an electric current. Those which conduct are called electrolytes, those which do not, nonelectrolytes. Knowledge of the electrical properties of dis- solved substances has been of the greatest value in the study of colloidal solutions, hydrogen-ion concentration and other im- portant fields.
H-ion Concentration. — It is a well known fact that acids dissolved in water and dissociating into their ions give off hydrogen-ions. The extent to which an acid is dissociated de-
24 PHYSIOLOGICAL CHEMISTRY
termines its so-called ''strength". Strong acids are much dis- sociated, giving off much hydrogen ion; weak acids are but slightly dissociated, giving off little hydrogen ion. Even water is a weak acid (and a weak base) since it dissociates thus
+ - + -
HgO == H + OH. In water the numbers of H and OH ions are equal, and such a solution is called neutral. If the hydrogen ions exceed the hydroxyl ions in number, the solution is called acid. If the hydroxyl ions exceed, it is called alkaline.
The extent to which a substance dissociates .into its ions can be expressed by a figure called the dissociation constant. For
HgU
But for water or an aqueous solution the concentratian of undissociated HgO is so great that it can be regarded as a con-
+ - stant, and the equation thus becomes H X OH = Kw. Kw is
called the water constant. It has been found that there are
0.000,000,1 gram ions of hydrogen ions and of hydroxyl ions in
1 liter of water. Expressed with logarithms this figure is lO'^.
The above equation then becomes 10'^ X 10"^ = Kw = 10". If
we measure the concentration either of hydrogen ions or of
hydroxyl ions it is thus possible to calculate the other from this
equation.
The symbol used to express hydrogen-ion concentration is Ch- It indicates the amount of hydrogen ion in 1 liter of the liquid. Thus the Ch of water is 0.000,000,1 or 10 ^ It is obviously in- convenient to use such an expression, so a shorter symbol has been devised, the pH. The pH is the logarithm of the Ch, with the minus sign omitted. Thus the pH of water is 7. If the solution is acid, the amount of hydrogen ion wdll be greater than lO"^, say 10 ^ 10"^, etc., and the pH will be 6,5, etc. A pH smaller than 7 indicates thus that the liquid is acid in reaction, whereas in an alkaline solution the Ch is less than lO-'', say 10-^, 10-®, etc., and the pH will be 8, 9, etc., that is, greater than 7.
PHYSICAL CHEMISTRY 25
Titratable Acidity. — It often is desirable to know the total amount of an acid or alkali present in a solution, regardless of the extent to which it is dissociated. The standard used in dis- cussing this value is the normal solution. A normal solution of an acid is one which contains per liter one gram equivalent (1.008 grams) of replaceable hydrogen. This hydrogen need not be ionized, but only ionizaUe. If 36.458 grams of hydrochloric acid is dissolved in water and made up to 1 liter, this will contain 1.008 grams of ionizable hydrogen, and will be a normal solu- tion. Any decimal fraction of this strength also may be pre-
N pared as --rr (0.1 N), etc. If the acid is sulphuric, one-half
the molecular weight will be used, as it will furnish 1.008 grams
of replaceable hydrogen. This is more fully discussed, and
directions are given for making up normal solutions in the
laboratory section. A normal solution of an alkali is one which
will neutralize volume for volume a normal solution of an acid.
If the acid were completely ionized, it would be easy to com-
N pute the pH of any normality. Thus, in a-r^ acid there is 0.1
gram ions of hydrogen per liter. The Ch is 10"^ the pH is 1.
N To calculate the pH of a-y^ alkali one falls back on the water
+ — — 20-1*
constant H X OH == lO^^ If H == lO-i then OH = -^ = 10^^
N The amount of hydrogen ions in a -r-^r- sodium hydroxide solu- tion is thus 0.000,000,000,000,1 or lO'^^^ and the pH is 13. This
+ -
is evident, for if the equation H X OH = 10-^* holds true for
+ -
water solutions, an increase in either H or OH will of course
N cause a decrease in the other (the figures given for -r^ acid
and alkali assume complete dissociation, which really does not
26 PHYSIOLOGICAL CHEMISTRY
take place at these concentrations, but the pH figures given are approximately correct for HCl and NaOH and illustrate the point without undue complexity).
A H-ion concentration of 0.035 per liter may be expressed as 3.5 X 10 ■^ etc.
From a biochemical viewpoint the hydrogen-ion concentra- tion is of the very greatest importance. On it depend the action of enzymes, the behavior of colloidal solutions (see later dis- cussion) the proper functioning of cells and tissues and other important processes. The determination of the hydrogen-ion concentration can be made accurately by electrochemical meth- ods, but these are too difficult for the average physician. It is possible to make approximate determinations by the use of sub- stances called indicators. These are compounds of various types which have one color in acid solution, another in alkaline. Their use in titration already should be familiar to the student. Now it happens that the indicators differ in the point of acidity (pH) at which they change color. Some change at the neutral point (pH = 7) others at a pH of 4, 5, 6, 8, 9, etc. The reason for this is as follows. The common indicators are themselves weak acids. As the free acid they have one color, as their salts another color. This is believed to be due to an internal rear- rangement of the molecule when in the salt form. Some indi- cators are stronger acids than others. If an alkali is added to a mixture of two acids, one strong and the other weak, the strong acid gets the base and forms its salt. If more alkali is added, finally a point is reached at which the weaker acid begins to get some of the base and form its differently colored salt, and a color change occurs. The weaker the acid (the indicator) the more alkali will have to be added before it can get any of the base. Two examples will illustrate this, methyl orange and phenolphthalein. Of these, the former is the stronger acid. When, by the addition of alkali the pH of an acid solution has been brought to 3 or 4, the methyl orange will get some of the base and change color. The weaker acid phenolphthalein, will
PHYSICAL CHEMISTRY 27
not get any of the base until the pH has been brought to 9 or 10. If one wishes to titrate a weak acid, such as an organic acid, one will choose phenolphthaiein as indicator, for the weak acid gives so low a hydrogen-ion concentration that on the addition of alkali the turning point of such an indicator as methyl orange (pH 3 to 4) would be reached long before the acid had been neutralized. On the other hand, if one wishes to titrate a strong acid (HCl) in the presence of a weak one (HgCOg or an organic acid) it will be possible to do so using methyl orange, for even a small amount of HCl gives a concen- tration of H ions high enough to affect methyl orange, whereas, when the strong acid is all neutralized, the methyl orange changes color even though there is a weaker acid still present untitrated.
Of course such values are not strictly accurate, but they are sufficiently so for many biological purposes.
Also the hydrogen-ion concentration of a solution can be de- termined by the indicator method. By making up a series of solutions of graduated hydrogen-ion concentration and adding certain indicators, one can get a series of solutions graded in color or depth of hue. Making up an unknown solution under similar conditions and adding the indicator, it is possible to match the hue with that of one of the tubes in the standard series, and thus determine with a fair degree of accuracy the pH of the unknown.
The student should be careful to get clearly in mind the difference between hydrogen-ion concentration and titratable acidity. The former is a measure of the amount of dissociated or ionized hydrogen per liter; the latter is the amount of re- placeable hydrogen, which need not necessarily be ionized.
N Whereas the titratable acidity of ——- solutions of hydrochloric
and acetic acid is the same, assuming of course the use of an indicator sufficiently sensitive to H ions, the former has a far greater H-ion concentration than the latter, because the former is more highly dissociated.
28 PHYSIOLOGICAL CHEMISTRY
Substances called buffers are of great importance in biochem- istry. If one adds a small amount of hydrochloric acid to water, the water becomes distinctly acid, and its pH changes from 7 to some smaller figure. For example 1 liter of water (or say 999 c.c.) (pH = 7) is acidified with 1 c.c. 0.01 HCl (pH about 2). The acid is diluted 1000 times. The Ch of the solution now
^T(ioff -^^^'^1 ^^^ t^® PH = 5. This small amount of dilute
acid has caused a marked change in the pH. Suppose that acid
N N
were added to a solution of — NaHCOg. If a whole liter of --
HCl were added to a liter of the bicarbonate, the NaHCOg would be converted into HgCOg, which is a very weak acid and the solution would still be somewhat near to the neutral point. The NaHCOg, itself forming a solution which is about neutral, still can neutralize strong acids, and the solution remains near the neutral point until the NaHCOg has been all used up. NagHPO^ and NaHoPO^, proteins, and various other substances behave similarly, and are of the greatest importance as buffers maintaining the reaction of blood and tissues near the neutral point even when acids are produced or absorbed.
Colloidal Solutions. — Certain types of substances when dis- solved in water form solutions which differ in many respects from solutions of ordinary chemicals such as hydrochloric acid, sodium chloride, sodium hydroxide, etc. The solutions often are opalescent, they may set to solid jelly like masses, the dissolved substance will not diffuse through a parchment membrane, and they show various other distinguishing reactions. Graham gave the name ''colloids" to such substances to distinguish them from substances which form "true solutions." These latter he called crystalloids. Since many of the constituents of living tissues form colloidal solutions the properties of such solutions are of great importance in biology, and have been much studied. It soon became evident that the colloidal state is only a condi- tion in which any substance can exist, if properly prepared, and is not confined to "glue-like" substances (colloidal means
PHYSICAL CHEMISTRY 29
''like glue")- Substances which usually are colloidal often can be 'crystallized, whereas crystalloids also can be made to assume colloidal form. It has become known that colloidal properties are dependent on the fineness of subdivision of the dissolved substance, that is, the size of the particles in the solution. If a more or less coarse insoluble material is shaken up with water, a portion remains suspended. This mixture is called a suspen- sion. If the particles of this suspended material could now be ji:round up so fine that they no longer could be seen, even with the aid of a microscope, we would have a cloudy liquid, the particles of which would not sink to the bottom on standing. If the particles were sufficiently small, we now would have a colloidal solution. If this process were continued further, we would ultimately arrive at individual molecules. This would then be called a true solution, since the dissolved particles now would be so small that they could pass through the pores of a parchment membrane. Fairly definite limits have been set for the size of colloidal particles. 1 /x is 0.001 mm. and 1 ft/x is 0.001 /x or one-millionth of a millimeter. The size of colloidal particles has been set as ranging from 1 to 100 fifi.
In thus subdividing material into such extremely small par- ticles the surface is enormously increased. If a cube of material 1 cm. on edge were divided into cubes 10 fx/x on edge the surface would be increased from 6 sq. cm. to 600 sq. meters and there would be 10^^ particles. The peculiar properties of colloidal solutions are thus largely dependent on the enormous increase of surface, and the consequent importance of surface forces, such as surface tension.
Classification and Properties of Colloids. — The colloids are by no means a unified chemical group, for substances of the most widely diverse chemical nature, such as metals, salts, acids, bases, proteins, carbohydrates, etc., may form colloidal solu- tions. The term ''colloidal" refers in fact to a state of mat- ter, and not to a class of compounds. Many substances of the greatest biological importance form colloidal solutions, in fact the constituents of the living cell are believed to be in a col-
30 PHYSIOLOGICAL CHEMISTRY
loidal state, so that the properties of colloids are both inter- esting and important. The group is divided into two classes, hydrophile or emulsoid colloids, and suspensoid colloids. Hy- drophile colloids more closely approach the crystalloids in their properties, whereas suspensoid colloids are more nearly like suspensions. As a matter of fact there is no sharp dividing line either between the groups, or between emulsoids and true solutions, or suspensoids and suspensions, since all gradations exist. Peptones, although belonging to the class of derived pro- teins, will pass through a parchment membrane fairly well, and are thus between the emulsoids and crystalloids. Certain metal hydroxides form gels, but are precipitated easily by electro- lytes, and are thus between the emulsoids and suspensoids. KaoJin shaken up with water is midway between the suspensoids and true suspensions.
Emulsoid colloids are characterized by the fact that they form gels if sufficiently concentrated, and are not easily pre- cipitated by the addition of salts. If in solution, the substance is called a hydrosol, if in a gel form, a hydrogel. Examples of emulsoids are albumin, gelatine and other proteins, starch, etc. The emulsoid colloids have some attraction for, or relation with the water surrounding them. This property is of the greatest importance, for the colloids of the living protoplasm aid in holding the water which is essential to the life and functioning of living cells.
The suspensoid colloids do not form gels, and are easily pre- cipitated by the addition of even a small amount of a salt. Ex- amples of this class are colloidal metals, sulphides, etc. They seem to have little relation to the water surrounding them.
The particles in colloidal solutions are so small that they will pass through an ordinary filter with ease. Filters impregnated with collodion have been prepared, however, by means of which colloid particles can be held back. In this, and other ways, the size of the particles has been estimated.
Tyndall's Phenomenon. — Colloidal solutions show an Inter- esting behavior known as Tyndall's phenomenon. If a beam
PHYSICAL CHEMISTRY 31
of light is passed through a colloidal solution, the path of the ray becomes visible, in much the same manner as the path of a ray of sunlight in a dusty room. The light is dispersed or re- flected from the particles of the colloid.
An instrument devised on the above principle is known as an ultramicroscope. While the observer looks through a high power microscope at a drop of the solution, a powerful beam of light from an arc is passed through the solution fro,m the side. The observer sees tiny flashes of light reflected up from the col- loidal particles. Only those of larger size will show this phenomenon, as colloidal gold particles. Colloidal albumin is invisible in the ultramicroscope. The limit of microscopic visi- bility is about 0.1 /x. Particles of this size or larger are called microns. The limit of ultramicroscopic visibility is about 15 fifx. in electric light or 5 /x/x in bright sunlight. Particles ranging from 1-100 fifjL are called submicrons, and those smaller than 1 fijuL amicrons. Molecules and ions are much too small to be visible in the ultramicroscope. As seen in the ultramicroscope, colloidal particles appear to be jumping about rapidly. This is known as Brownian movement, and is supposed to be due to the bombardment of the particles by the molecules of the solvent.
Electrical Properties of Colloids. — Colloidal particles carry electrical changes just as ions are electrically charged. This may be demonstrated by passing an electric current through a colloidal solution. The particles of the colloid will move to the positive or negative pole according to the nature of the charge carried, — a colloid with a negative charge travelling to the positive pole, and vice versa. This phenomenon is known as cataphoresis. Whereas some colloidal particles probably have but one electrical charge, undoubtedly they often carry more than one. A protein in colloidal solution will have a positive charge if the solution is acid in reaction, but a negative charge if the solution is alakaline. We may imagine that this is brought about as follows : in acid solution the protein combines with some of the acid, for example hydrochloric acid. From this complex compound, negatively charged chlorine ions are
32 PHYSIOLOGICAL CHEMISTRY
given off into the water, and positive charges will remain on the colloid particles. In alkaline solution the protein forms salts, such as the sodium salt. Sodium ions are given off, carry- ing positive charges, and negative charges will remain on the colloid particles. These facts are of great importance in many of the precipitation reactions of the colloids.
Methods of Precipitating Colloids. — Some colloidal solutions will precipitate, the colloid flocking out, merely on standing. Some will precipitate if they are boiled. Some substances are soluble in hot water, but their solutions will solidify on cooling. Some colloids are thrown out of solution by the addition of an electrolyte. The suspensoid colloids are precipitated by adding a ve^y small amount of an electrolyte such as a salt or an acid, but the emulsoid colloids are precipitated much less readily, that is, only by adding much more of the electrolyte. It has been observed that the effective part of the precipitating salt or substance is the ion bearing the opposite charge to that on the colloid. If the precipitating part of the salt is the metal, then in general colloids bearing negative charges will be precipitated. In this connection it has been observed that trivalent metals are better precipitation reagents than divalent metals, and divalent metals in general are better precipitation reagents than mono- valent metals. Thus to precipitate a given colloid from solu- tion a ferric salt is better than a mercuric salt, and a mercuric salt better than a sodium salt. That is, a smaller concentration of ferric chloride than of mercuric chloride is required, etc. But all ions of the same valence do not have equal precipitation powers. They vary according to their solution tension.
"When a colloid is precipitated by an electrolyte the precipi- tate contains some of the precipitating ion, so the precipitate is believed to be a compound of the colloid and the precipitating ion. The precipitation of colloids, however, is undoubtedly de- pendent on other and more complicated factors than the mere formation of salts or similar compounds of the colloids. For further discussion of this subject the student is referred to larger or more specialized works.
PHYSICAL CHEMISTRY 33
A suspensoid colloid, as has been stated, is easily precipitated by the addition of an electrolyte. If a small amount of an emul- soid colloid is added to a suspensoid colloid solution, the latter is much less easily precipitated. The suspensoid colloid is "pro- tected" by the emulsoid colloid (albumin, for example). This phenomenon is called the protective action of colloids.
Much interest has centered around gel formation and gel structure. Often a very small amount of material will form a gel holding fairly rigid a relatively enormous volume of water. The jelly like consistency of protoplasm is due to the colloidal character of its constituents, so that gels are of the greatest biological importance. Gels are now considered to be a net- work of tiny crystal-like formations which hold the water in large measure enmeshed. If a gel stands for some time, the mass of crystals shrinks, squeezing out some of the liquid. This process is called synresis or ''bleeding."
Emulsoid colloids enormously increase the viscosity of solu- tions, suspensoid colloids scarcely at all.
Adsorption — Surface Tension. — Colloidal particles have the property of taking up other substances on their surfaces. This process is called adsorption. It is of great biochemical interest, since it undoubtedly plays a part in many important processes, such as the formation of the temporary union between an en- zyme and its substrate, the combination of toxins with anti- toxins, the sensitization of leucocytes by opsonins, the taking up of bacteria by leucocytes, the formation of compounds be- tween proteins, lipins and other constituents of the cell.
The process is closely related to surface tension.
At the surface between two media, for example water and air, the molecules of water in the surface layer are attracted down- wards and to all sides by their fellows, but not up. The mole- cules in the surface are thus drawn down, and form what is called the surface film. There is a constant tendency for the amount of free energy in a system to decrease. This is the principle of Willard Gibbs. Now in the surface between two liquids, or a liquid and a solid, the presence of dissolved sub-
34 PHYSIOLOGICAL CHEMISTRY
stances tends to decrease the surface tension. Thus, in accord- ance with Gibbs' principle the dissolved substance will tend to collect or accumulate in the surface film, — to concentrate on the surfaces of the dissolved particles. This is adsorption. Of course the adsorbed substance may combine chemically with the material in colloidal form, and it may be held on by elec- trical forces. Adsorption phenomena are without doubt of great importance in the body. Much study is being expended upon them at the present time.
Imbibition. — The swelling up of a substance like gelatin when placed in water is called imbibition. The gelatin particle in- creases greatly in size, but does not go into solution. This process is important, as in this way cells take up water. The extent to which imbibition will proceed is greatly influenced by the presence of salts, acids, bases, etc. Salts usually repress the swelling to a point below that which would be reached in pure water. Acids affect it differently according to their con-
N centration. At about oHR the greatest swelling takes place. If
the strength of the acid is less than this, the swelling will be smaller than in pure water. In stronger acid, the swelling is at first rapid, but it does not reach so great a final value as in the concentration specified above.
From the foregoing pages it will be evident that colloidal solu- tions are of great interest in connection with the study of the chemistry of the body.
CHAPTER II ELEMENTS, INORGANIC MATERIALS, WATER
Elements Found in the Body. — The body is made up of a large number of chemical elements which are present in very unequal amounts, and distributed quite unevenly in the vari- ous tissues and body fluids. Certain of these elements are in all living cells, others only in particular kinds of cells or in particular animals. Still others are present only accidentally or temporarily. The elements found most frequently are the non-metals carbon, hydrogen, oxygen, nitrogen, sulphur, and phosphorus ; the metals sodium, potassium, calcium, magnesium and iron; the halogens, chlorine, iodine, and fluorine. In addi- tion, there are many other elements such as silicon, copper, manganese, arsenic, silver, lead, bromine, lithium, etc., found only in traces or only in a few animals.
Importance Not Determined by Amount Present. — Three of the elements, carbon, hydrogen and oxygen alone make up over 90% of the body weight. The conclusion should not be drawn, however, that these are the only important elements and that those elements or compounds present in small amounts or traces are relatively unimportant to the organism. Quite the contrary may be the case. The body of an average-sized adult man contains only about three grams of iron, and yet this is so necessary to life that an animal fed for some time on a diet which contains no iron will die quite as surely, though of course not as quickly, as if it had been deprived of food alto- gether. The principle involved may be formulated as the Law of the Minimum which states that the importance of a given substance to the animal organism is independent of the rela- tive amount in which it is required. Striking examples of this principle have developed in recent years, and we now know that the body requires certain compounds of which nothing
35
36 PHYSIOLOGICAL CHEMISTRY
was known a few years ago. Some of these substances appear to be organic compounds. Although the amounts of these ma- terials of unknown constitution which are required by the body are only a small fraction of a gram, still their absence from the diet will cause severe disorders and ultimate death.
The importance to the body of the individual elements ex- tends much beyond the function of serving as inert building stones out of which the body materials are made up. Many of the chemical reactions in the body are greatly influenced or .modified by certain of the elements. For example, two important processes, the clotting of the blood which tends to protect a wounded animal from bleeding to death, and the clotting of milk in the stomach, the first step in its digestion, are dependent upon the presence, among other things, of calcium, and without this metal neither of these reactions can occur.
The accompanying table gives a survey in round numbers of the relative amounts of the elements present in the body.
Carbon 17.5
Hydrogen 10.2
Oxygen 66
Nitrogen 2.4
Sulphur 0.2
Phosphorus 0.9
Sodium 0.3
Potassium 0.4
Calcium 1.6
Magnesium 0.05
Iron 0.005
Chlorine 0.3
Iodine Traces
Fluorine Traces
Other elements Traces
Carbon, Hydrogen, Oxygen, Nitrogen, Sulphur and Phos- phorus.— Carbon has the property of forming a very large num- ber of compounds with hydrogen, oxygen, and sometimes sul-
ELEMENTS, INORGANIC MATERIALS, WATER 37
phur, phosphorus and other elements. These compounds are so numerous that a separate branch of chemistry is devoted to them under the name of organic chemistry. Thirty per cent or more of the body consists of organic material, or compounds of carbon with hydrogen, oxygen, etc. The importance of these compounds is so great that special chapters will be devoted to the important groups. The body receives its carbon compounds in the foods, the solid portion of which is very largely organic material. Aside from the organic components of the tissues, carbon is also found in inorganic form, chiefly as calcium car- bonate in bone, and as sodium bicarbonate in the blood. This latter compound is of great importance, since, although its solution is neutral, it has the power of neutralizing acids, thus protecting the tissues when acids are introduced into the blood either from the digestive tract or as the result of the breaking down of body materials. Carbon is eliminated from the body as carbon dioxide, given off through the lungs, or as more com- plex compounds such as urea and other materials excreted in the urine or the feces. If heated, organic compounds char, leaving a black residue of carbon.
Hydrogen and oxygen aside from being constituents of water, organic and inorganic compounds, also play more specialized roles in the body. Oxygen, which makes up about two-thirds of the entire body weight is taken up as a constituent of the compounds in the food and in the process of respiration. Hy- drogen, the presence of which in ionic form is the distinguishing characteristic of acids, plays an important part in the digestion of proteins by gastric juice. Hydrogen and oxygen are elimi- nated from the body chiefly as water and CO2 given off through the lungs, and as water and in organic compounds in the urine and feces. On heating organic materials they decompose, and the hydrogen and oxygen are given off as water and various other compounds.
Nitrogen, sulphur and phosphorus also play various roles in the body and all three are found both in organic and inorganic compounds. Nitrogen is a constituent of all proteins. Many
38 PHYSIOLOGICAL CHEMISTRY
proteins contain sulphur and phosphorus as well. Nitrogen occurs in various other compounds throughout the body, and the body fluids contain gaseous nitrogen in solution, as is to be expected since nitrogen is somewhat soluble in an aqueous liquid, and in the lungs the blood comes in contact with the air which is made up about four-fifths of nitrogen. So far as is known this dissolved nitrogen has no influence on the body's activities. Nitrogen is excreted mainly as urea, uric acid and other products formed by the decomposition of proteins in the body. If heated with soda lime, an organic substance of this type gives off its nitrogen as ammonia. Sulphur and phos- phorus are present in various compounds other than the pro- teins. Of these the phosphatids are perhaps most interesting. This group will be considered later. Calcium phosphate makes up about 85% of the ash of bone. Sodium phosphate is found in the blood and tissues. Sulphur and phosphorus are excreted mainly in the urine as sulphates and phosphates, but also in the feces. Unoxidized sulphur may be split off from organic com- pounds by boiling with alkali. On adding lead acetate, a dark precipitate of lead sulphide will form. This test may be con- firmed by adding an acid. Hydrogen sulphide will be given off and may be identified by a paper moistened with lead acetate on which lead sulphide will form as a shiny dark precipitate. Sulphates are detected by precipitating as barium sulphate. Phosphates are detected by precipitating as ammonium phospho- molybdate.
Sodium, Potassium, Calcium, Magnesium and Iron. — These metals make up only a small part of the body, but they are none the less important. They are distributed widely throughout the body tissues and fluids. There usually is more sodium than potassium in body fluids, and more potassium than sodium in the solid tissues. These metals are present as chlorides, sul- phates or phosphates. Sodium chloride in the blood is interest- ing since it furnishes chlorine for the hydrochloric acid of the gastric juice, whereas sodium bicarbonate is very important in preserving the neutrality of the blood as above noted. Sodium
ELEMENTS, INORGANIC MATERIALS, WATER 39
chloride is the only inorganic substance which is contained in a mixed diet in amounts insufficient for the body's needs. Ac- cordingly our food must be salted. Herbivorous animals, living on plants in which potassium is in excess of sodium, crave salt, and it is well known that cattle must be "salted" to keep them in good health. Sodium and potassium also affect the irritabil- ity of muscle and nerve tissue so that their role in the organism may perhaps include little understood regulatory functions. These metals may be detected by the flame test or by precipita- tion as sodium pyroantimonate or potassium cobaltinitrite.
Calcium and magnesium, while found in all cells in the body, are present in largest amount in the bones and teeth, which contain about 99% of all the calcium and 70% of all the mag- nesium in the body. Their phosphates and carbonates make up about 98.5% of all the inorganic material in bone. That these metals have other roles to play in the body, however, is demon- strated by the failure of blood and milk to clot in the absence of calcium. Calcium is detected by precipitation as calcium oxalate. Magnesium is present in much smaller amounts than calcium, and less is known of its physiological activities. It is interesting in this connection that magnesium sulphate acts as an anaesthetic on mammals and that it paralyzes the endings of the motor nerves in the muscles. . Magnesium is detected by precitation as magnesium ammonium phosphate.
Iron, though present only to the amount of a few grams in the body of an adult man, still is distributed very widely. Its most conspicuous role is in connection with the hemoglobin of the blood, of which it is a constituent. This substance carries oxygen from the lungs to the tissues. Iron probably is pres- ent also in traces of inorganic iron compounds. The spleen contains relatively much, so that it has been suggested that the spleen has some role to play in connection with the iron com- pounds of the blood. Also the liver is concerned with the fate of hemoglobin, and serves as a clearing house through which hemoglobin from worn out corpuscles is broken down and ex- creted by way of the bile into the intestine. Iron is also ex-
40 PHYSIOLOGICAL CHEMISTRY
creted in the urine, the amount being no greater than 10 or 11 mg. per liter.
Iron is detected by precipitation as ''Prussian Blue" or fer- ric f errocyanide or by the formation of ferric thiocyanate which is red in color.
Chlorine Iodine, Fluorine, etc. — Chlorides are present in traces in all the tissues and fluids; the blood contains 0.5-0.6% sodium chloride, the gastric juice about 0.4% hydrochloric acid. The necessity of adding sodium chloride to the diet has already been referred to. Chlorides are excreted in the urine in amounts varying with the amount in the food, 10-12 gms. daily being an average figure. Chlorine may be detected by precipitation as silver chloride. Iodine is interesting chiefly in connection with its occurrence in the thyroid gland. This gland, by means of a compound which it produces and pours out into the blood stream, has far reaching influence on the chemical reactions going on in the body. This important substance is an iodine compound. If an iodide gets into the blood stream, the salivary glands have the power of excreting it. After taking a capsule containing potassium iodide, iodine may be demonstrated in the saliva. Fluorine is found in the bones and teeth.
Other elements may occur either regularly or accidentally in the body tissues or fluids, but usually only in traces.
Water. — Water makes up about % of the body weight of mammals, and a much larger part in some lower animals. All tissues contain it, even the enamel of the teeth: blood, lymph, the digestive juices, urine, etc., are %o-%o water; the organs and softer tissues about %. Water serves a variety of func- tions. It is the circulating medium for transporting food and waste material to and from the cells, it holds various sub- stances in solution and makes ionization possible, and is a medium for the excretion of waste. It also distributes the heat of the body, and by evaporation from the skin is instrumental in regulating body temperature. An animal may survive for many days without nourishment, but if water also is withheld, death follows in a few days' time.
CHAPTER III
CARBOHYDRATES
Composition, Occurrence, General Function. — The carbohy- drates are compounds of carbon, hydrogen and oxygen in which the hydrogen and oxygen are present usually in the same pro- portions as in water, hence the name of the group. A few carbohydrates do not conform to this general statement, for example Rhamnose CcHigOg. Some substances not carbohy- drates do contain hydrogen and oxygen in this proportion, such as acetic acid CgH^Og and lactic acid CgHgOg. Thus, this char- acteristic is not distinctive of the group. Carbohydrates vary considerably in their properties. They are found both in plants and in animals, but chiefly in the former, in which they form a considerable part of the structural frame work. In animals they serve mainly as a fuel to be oxidized for the production of heat or the performance of mechanical work, but they may be laid away as a reserve store to be called on in case of need.
Structure of the Carbohydrates. — The carbon atoms in the carbohydrate molecule are linked together in a long chain. It has been shown that the molecule contains several hydroxyl groups, and that in the simpler members of the group at least
H
I I
there is either an aldehyde — C = 0 or a ketone C = 0 group.
I I
The remaining valencies of the carbon atoms in the chain hold the hydrogen atoms not accounted for. Thus the formula for glucose, one of the most important carbohydrates, has been shown to be as follows:
41
42 PHYSIOLOGICAL CHEMISTRY
H
I
I ■ H— C— OH
I HO— C— H
I H— C— OH
I H— C— OH
I CH2OH
Glucose
An example of a sugar containing a ketone group is fructose, which has the following formula:
|
CH2OH |
|
|
1 C=0 1 |
|
|
HO- |
1 -C— H 1 |
|
H- |
-C— OH 1 |
|
H- |
-C— OH 1 |
|
CH.OH |
|
|
Fructose |
Many of the reactions characteristic of the carbohydrates de- pend upon the properties of the aldehyde or ketone groups which they contain. In the case of the more complex carbo- hydrates, these reactive groupings are usually combined in such a way that they do not show their characteristic behavior.
Optical Activity. — On inspecting the above formulas for glucose and fructose it will be observed that the hydrogen and hydroxyl groups of the four carbon atoms occupying the middle portion of the chain are not arranged in a regular manner.
CARBOHYDRATES 43
This irregular arrangement is intended to indicate that there is actually a variation in the arrangement of these groups around the carbon atoms in space. This arrangement is a determining factor in the property of these substances known as optical activity. Optical activity is the property possessed by many compounds of rotating the plane of polarized light.
If a ray of white light is passed through a crystal of Iceland spar it is split into two slightly diverging rays, but this is not the only change which is produced in the ray. Light is due to vibration of the particles of the ether, the vibration being at right angles to the direction of the ray, and in all possible planes passing through the path of the ray as an axis. We can picture this perhaps by imagining the cross-section of a ray of light to resemble the cross-section of an orange, only with many more planes of vibration. After passing through the crystal of Iceland spar, the light is so altered that in each of the two em^erging rays vibration is taking place in only one di- rection. To light of this character is given the name plane polarized ligJit. It has been found that optically active sub- stances have the property, if such a ray of light is passed through their solutions, of rotating the plane in v/hich the ether particles are vibrating, some substances rotating the plane of vibration to the right, others to the left. Not only do these compounds possess this property, but a given substance, under similar conditions of observation (concentration, temperature, etc.) always rotates the plane of polarized light through the same angle. This uniformity of behavior on the part of each optically active substance gives us a most useful means of de- tecting the presence of the compound in question.
In order to have a uniform standard for comparing observa- tions of optical activity it is necessary to adopt some system for reporting such data. A value has been selected and given the name ''Specific Rotation," which is the rotation produced by 1 gram of substance dissolved in 1 cubic centimeter of sol- vent and the rotation observed in a tube 1 decimeter in length. This value is designated by the symbol [ oc ] .
44 PHYSIOLOGICAL CHEMISTRY
It is obvious that it often will be impossible to observe the rotation produced by a substance under these standard condi- tions. Many substances are not sufficiently soluble to dissolve 1 gram in 1 cubic centimeter of solvent. To avoid this difficulty, a formula has been developed for use under general laboratory conditions. Let oc be the observed rotation of a solution under question. Under standard conditions [ oc ] == oc . Let g = grains substance per c.c. of solvent. If g is not equal to 1, we can
easily find the value of [ oc ] by dividing oc by g. [ oc ] = — '-
We must also introduce the length of the observation tube, as naturally a column of liquid longer or shorter than the specified 1 decimeter will give respectively a greater or a smaller rota- tion. Our formula will now be [ oc 1 =— -^
l.g
As it seldom is convenient to work with 1 c.c. of solution, it will be advisable to revise our formula for use with solutions calculated on the basis of 100 c.c. Let c equal the number of
c
grams substance in 100 c.c. solvent, then g = y^ Substituting
this value of g in the above equation we have
. , 100. ex
Now the amount of rotation which a given solution will pro- duce is influenced by various factors, among them the tempera- ture of the solution, the color of the light used in the observa- tion, etc. Unless there are special reasons for other procedures, it is customary to make observations at 20° C. and with sodium light, corresponding to the D line in the spectrum. These two influencing factors also are included in our formula, which we now have in its final form.
, , 20° 100. oc
D l.c
This value is called the specific rotation of a compound and is a constant for each optically active substance. The specific rotations of most of the sugars have been determined, and may
CARBOHYDRATES 45
be found in textbooks or books of reference. By observing the rotation cc of a solution of a known sugar and substituting this value in the above equation it is possible to calculate the amount of the given sugar present. On the other hand, if we have a solution containing a known amount of an unknown sugar, it is possible to calculate its specific rotation, which usually will identify the substance, especially if used in conjunction with other tests. For this purpose we may solve our formula for c
100. PC ^ ~1. [ oc ] 20° D The actual observation of oc is made with a polariscope, an instrument in which light is polarized and passed through the solution to be examined. The apparatus is so constructed that it is possible to measure the amount of rotation produced by the solution. The accompanying diagram indicates the struc- ture of a Laurent polariscope.
]^
d e f
Fig. 1. — Diagram op Laurent Polariscope.
A. Source of light, — sodium flame.
B. Plate cut from a crystal of potassium bichromate. In place of this, a flat-sided cell
containing a solution of potassium bichromate may be used to insure absence of other than yellow light.
C. Lens to make the rays of light parallel.
D. Nicol prism, called the polarizer, which polarizes the ray.
E. Quartz plate covering a portion of the field to produce half shadow
F. Tube containing solution to be studied.
G. Second Nicol prism called the analyzer. H. Lenses for focusing.
1. Eye of observer.
The Nicol prism is a device for polarizing light, and getting rid of one of the two rays into which the original ray is split. A crystal of calcite is sawed through diagonally, and the two pieces stuck together with a thin layer of Canada Balsam.
On entering the prism, light is polarized and split into two diverging rays. From the diagonal surface, one of the two polarized rays is reflected to the side, the other passes on through
46 PHYSIOLOGICAL CHEMISTRY
the apparatus. If the tube / contains only water, the polarized vixy i)asses through it without altering the direction of its vibration. On arriving at the second Nicol prism g the ray wiJl pass through unaltered provided the prism is in a posi- tion corresponding to that of the first prism. If, on the other hand, the prism g has been rotated to the right or the left so that its position no longer corresponds to that of d, only a por- tion oE the ray will pass through, and the intensity of the ray j'caciiing the eye at i will be diminished. If the prism g is rotated 90° from the position corresponding to that of d, no liglit will pass through. Beyond this point the illumination in- creases until at 180° it again is at its maximum. At 270° the field again is dark.
Imagine the apparatus set with the two Nicol prisms in cor- responding positions. Light will pass through to the eye. Now if a tube of sugar solution is placed at /, the ray of light passing from d will be rotated about its axis by the sugar solu- tion, which is optically active. The result will be that it strikes g in a position in which it will not pass through without losing some of its intensity. If we rotate prism g through the same angle through which the ray of light has been rotated by the sugar solution, the ray again will pass through at maximum intensity. By observing the angle through which the prism must be rotated to bring this about, the angle of rotation of the ray caused by the sugar solution is determined.
In practice it would be difficult to observe a changing illu- minated field and select the point at which the illumination was at its maximum. A mechanism has been devised to obviate this difficulty. This is represented in the diagram by e which is a thin quartz plate covering one-half the visible field. This plate so alters the light passing through it that when the half of the field not covered by the plate is at its maximum illu- mination, the half of the field covered by the plate is somewhat darker, and vice versa. By rotating the prism g a point can be found intermediate between the two, at which the two halves of the field are equally light. This is taken then as the
CARBOHYDRATES 47
starting (or zero) point of an observation. When the sugar solution has been introduced the prism g is rotated until the two halves of the field again are equally light, and the reading taken. This will correspond to the rotation produced in the polarization plane of the ray by the sugar.
The readings are made upon a circular scale which usually is graduated in degrees, and provided with a vernier to make it possible to read to minutes.
The rotation produced by a given substance varies with the solvent. Water is the solvent most frequently used. If more than one optically active substance is present in the same solu- tion, account must be taken of this fact, or one or the other removed. Certain sugars when first dissolved in water show a much higher or much lower rotation than after standing for twenty-four hours or so. This is believed to be due to the fact that these substances exist in two forms, of which one possesses much stronger rotating power than the other. The two forms pass into one another spontaneously, and reach an equilibrium in which there is a definite amount of each, the rotation pro- duced by this final mixture being the resultant of the rotations of the two forms present. To this phenomenon is given the name Mutarotation (from the Latin word meaning ''to change"). Some sugars show about twice the final rotation when first dissolved, e.g., glucose, and for such cases the term birotation may be employed. Half rotation is the term employed for tliose substances showing half the final rotation when first dissolved. Instead of allowing a solution to stand until equilib- rium is reached, this may be accomplished at once by adding a small amount of alkali to the solution.
The exact reason why some compounds have the property of rotating the plane of polarized light is unknown. This property has been shown to depend, however, upon the presence of what has been called an asymmetric carbon atom, which is a carbon atom united to four dissimilar chemical groups. If the struc- tural formula for glucose is inspected, it will be seen that in the case of four of the carbon atoms in the chain each is united
48 PHYSIOLOGICAL CHEMISTRY
to four different chemical groupings. Each of these four car- bon atoms is thus asymmetric, and confers the property of opti- cal activity upon the compound. Around each asymmetric atom either of two groupings may exist,, one of which rotates the plane of polarized light to the right (dextro-rotatory), the other an equal distance to the left (levo-rotatory). These two compounds are called optical isomers. It has been found that the number of optical isomers possible when a compound con- tains more than one asymmetric carbon atom, is represented by the value of 2° where n is the number of such carbon atoms. We will thus expect to find 2*=16 different sugars, all isomers of glucose. Twelve of these sixteen actually have been prepared. Classification of Carbohydrates. — The carbohydrates are divided into three great classes.
These are subdivided as indicated below. Monosaccharides Bioses Trioses Tetroses
Pentoses — arabinose, xylose, ribose Hexoses — glucose, fructose, galactose, mannose Heptoses Octoses Nonoses
Disaccharides
Saccharose (sucrose, or cane sugar) Maltose (malt sugar) Lactose (milk sugar)
Polysaccharides Dextrins Starches Glycogen Inulin Cellulose Gums and Mucilages.
CARBOHYDRATES 49
The monosaccharides are sometimes called the simple sugars. The disaccharides are so called because they are formed by the union of two molecules of monosaccharide, with elimination of water. Polysaccharides are composed of several molecules of monosaccharide united in a similar manner. The monosac- charides are subdivided into groups according to the number of carbon atoms in the molecule, thus the bioses are sugars hav- ing only two carbon atoms in each molecule, the trioses, three, etc. The first six of these groups are found in nature. The octoses and nonoses have been built up in the laboratory. Of the monosaccharides, the pentoses and hexoses are the most im- portant. With the exception of the bioses, there are two classes of sugars in each group, — aldehyde sugars and ketone sugars. Of the biologically important sugars all are aldehyde sugars, or aldoses, with the exception of fructose, which is a ketone sugar or ketose. The individual carbohydrates will be discussed later.
Origin and Synthesis. — The ultimate dependence of the animal world upon plants is well illustrated by the carbohy- drates. These compounds which are an important fuel for the body are obtained from plants, in which they are built up from the very simple substances carbon dioxide and water. The exact mechanism by which the plant brings about this important synthesis is a matter of some uncertainty, but it is probable that the carbon dioxide from the air is first reduced to formaldehyde. Several molecules of formaldehyde then condense to form a carbohydrate. The reaction might be represented as follows:
CO^-fH^O-^H-CHO-fO^ 6 HCHO-^CeHi^Og
The energy for this synthesis is derived from sunlight by the agency of chlorophyl, the green coloring matter of plants. The hexose so formed may then be built up into more complex substances such as starch, which is laid away as reserve food in the seeds, tubers and other parts of the plant. Sunlight is not necessary for the second part of this process, as starch can be built up in the roots and tubers, which are underground. By
50 PHYSIOLOGICAL CHEMISTRY
variations of this. general process the plant undoubtedly can build up a large number of other compounds of the most diverse nature.
The animal body has much more limited powers of synthesiz- ing carbohydrates, although we now know that it is capable of doing much more in this respect than was once thought. The polysaccharide glycogen is regularly built up in the animal body from monosaccharides, and it has been shown in diseases where carbohydrates are lost from the body in the urine, that certain compounds other than carbohydrates apparently can be converted into sugar in the body.
There are various methods for synthesizing carbohydrates in the laboratory. One of these suggests the synthesis in plants. If a solution of formaldehyde is made slightly alkaline, a con- densation takes place, and the liquid will be found to contain a hexose, acrose.
A method which has proved very useful in studying the carbohydrates is known as the cyanhydrin synthesis. This serves to lengthen the carbon chain by one carbon atom. Start- ing from a pentose, a hexose may be prepared, from a hexose a heptose, and so on. The steps of the synthesis are as follows :
CH2OH— CHOH— CHOH— CHOH— CHO+HCN-» CH2OH— CHOH— CHOH— CHOH— CHOH^CN
This nitril, containing six carbon atoms is easily saponified.
HOH CHoOH— CHOH— CHOH— CHOH— CHOH— CN+HOH^
" HOH
CH2OH— CHOH— CHOH— CHOH— CHOH— COOH+ NH3+H2O
Converting this into its lactone by the action of acid we get:
CH2OH— CHOH— CH— CHOH— CHOH— C=0+H20
_l^— o 1
On reducing this with sodium amalgam the compound takes
CARBOHYDRATES 51
up 2H forming an aldehyde, a hexose, which thus has been built up from a pentose.
CH2OH— CHOH— CHOH— CHOH— CHOH— CHO
The carbohydrates also may be "built down" step by step in the laboratory. This method also has been of value in study- ing their constitution. If treated with hydrogen peroxide in the presence of a ferric salt as catalyzer, the elements of formic acid are split ofP, leaving a carbohydrate with one less carbon atom. Carbohydrates also may be built down by electrolysis or by way of their oximes.
Interconversion of Carbohydrates. — Since the different mono- saccharides are closely related compounds, differing only in the arrangement of their hydrogen and hydroxyl groups around the carbon atoms, it is not surprising to learn that certain of them may very easily be converted into one another. Thus if a solu- tion of glucose is made slightly alkaline and allowed to stand for some time, it will be found to contain not glucose alone, but also fructose and mannose. A portion of the glucose is transformed into the other two sugars. It is immaterial which of the three sugars is taken to start with, — the result will be the same and all three will be found in the solution. The inter- conversion of the simple sugars is of interest physiologically, for in the body such transformations are known to take place. The lactose of milk is produced in the mammary gland from glucose in the blood. Lactose is made up of equal parts of glu- cose and galactose. A portion of the glucose from the blood thus must be transformed into galactose in the gland. A second instance of a similar transformation is the formation of the polysaccharide glycogen which is stored up in the liver and muscles as a reserve material. On hydrolysis glycogen yields always glucose. It is well known, however, that glycogen will be deposited in the body if an animal is fed fructose or various other sugars. The glycogen formed under these circumstances also yields glucose on hydrolysis, indicating the transformation of the fructose, etc., into glucose.
52 PHYSIOLOGICAL CHEMISTRY
Combination of Carbohydrates With One Another, and With Other Substances. — The carbohydrates have the property of
combining with various substances including themselves. The union of two molecules of monosaccharide forms the disac- charides, of many molecules of monosaccharide, the polysac- charides. Compounds of monosaccharides with other classes of materials are variously named, and include substances of great biological interest and importance. Examples are the glucosides, — compounds of glucose or one of its derivatives, among which are many of the drugs used in medicine, and some of the constit- uents of brain and nerve tissue. The term ''Glucosides" is sometimes extended to include similar compounds which yield other sugars, e.g., galactose.
Behavior With Strong Alkalies. — The action of weak alkali upon the carbohydrates already has been discussed under the heading interconversion of carbohydrates. A rearrangement of groups in the molecule is observed. The action of strong alkali is much more vigorous and far reaching and the nature of the products formed depends upon the experimental conditions. The alkali undoubtedly combines with the sugar at first. There follows loss of water from groups around neighboring carbon atoms, and a double bond is formed. The carbon chain is then broken, and two compounds are formed, each with a smaller number of carbon atoms than the original substance. The nature of these fragments is only imperfectly understood, but it may be inferred from the final products of the reaction. In case oxy- gen is supplied plentifully, the fragments are oxidized to the corresponding acids. Of these a long list may be obtained vary- ing in complexity from formic H . COOH and oxalic acids
COOH to acids having
I COOH
chains as long or only slightly shorter than the chain in the original substance. Evidently the breaking up into fragments may take place at various points in the chain, giving compounds
CARBOHYDRATES 53
containing one, two, three, four, or five carbon atoms. The chain also may remain unbroken.
This process is of biological interest for probably it has many points of similarity with the breaking down of carbohydrates in the body. Of course the body tissues are not strongly alkaline, so that the agent bringing about the change must be some other substance. In the body the fragments thus produced may be used to construct new substances, or they may be further broken down and oxidized to carbon dioxide.
In case the strong alkali acts upon glucose when the oxygen supply is limited, as would be the case if no air were bubbled through the liquid, the reactive fragments produced will com- bine with one another, forming complex brown substances of a resinous nature, a mixture of which is known as caramel. Pos- sibly this process is analogous to the building up of materials from carbohydrate fragments in the interior of the body cells, although in this latter case the conditions of synthesis are care- fully controlled by agents in the cell so that particular sub- stances result which are required by the cell.
Behavior With Acids. — On boiling with dilute acids the com- plex carbohydrates take up water and are split into simpler substances, ultimately the monosaccharides. If the monosac- charides are boiled with strong hydrochloric acid they are de- composed and yield a variety of products. The pentoses lose water and form furfurol.
IFI H H IJil H
H C — C — C — C — C = 0 HC — CH
/ I -^ II II +3H,0
O HC C— CHO
E^ V
The formation of this compound serves to identify the pen- toses, since it forms with various phenols colored substances which may be identified easily. The hexoses, on boiling with concentrated hydrochloric acid yield among other things oxy- methyl furfurol, but in much larger quantities levulinic acid
54 PHYSIOLOGICAL CHEMISTRY
CH3 . C— CH2 . CH2 . COOH
II
0
Oxidation of Carbohydrates. — Oxidation Tests. — Since the simple carbohydrates contain either an aldehyde or a ketone group they are easily oxidized, even by mild oxidizing agents. The products of mild oxidation are acids having the same num- ber of carbon atoms as the original material. If the oxidation is more vigorous the molecule may break into fragments as was de- scribed under the action of alkalies. Some of the most important carbohydrate tests depend upon oxidation processes. Among the oxidation tests are the Fehling, Benedict, Almen-Nylander, and Barfoed tests.
Fehling Test. — If solutions of copper sulphate and sodium hydroxide are mixed, a light blue or whitish precipitate is formed. This is cupric hydroxide
CUSO4+2 NaOH^Cu(OH)2+Na2S04
If this mixture is boiled, the precipitate is converted into black cupric oxide.
Cu(OH)2-»CuO+H20.
If a sugar is present in the solution, however, the copper is reduced by the sugar which is converted into an acid. If a sugar is added to a mixture of copper sulphate and sodium hydrate the liquid will turn a very deep blue, and a much smaller precipitate will form as most of the Cu(0II)2 is held in solution by com- bining with the sugar. If the mixture is allowed to stand, or if it is boiled, the color becomes perhaps yellow at first, and ultimately a red precipitate forms. The sugar has reduced th^ cupric hydroxide to yellow cuprous hydroxide, which, on boiling, decomposes into the red cuprous oxide.
2 Cu(OH),+C5HiA.CHO-^2 CuOH+
C5H11O, . COOH+H2O 2 CuOH-^Cu^O+HoO
As a matter of fact, the sugar undoubtedly undergoes further change, as we already have seen in considering the action of al-
CARBOHYDRATES 55
kalies on the carbohydrates, but the above equations serve to show the nature of the. part played by the copper compound in the reaction.
The . above test would work very well if the solution to be tested contained much sugar. If this were not the case, how- ever, much black cupric oxide would be formed which might easily obscure any small amount of red cuprous oxide resulting from the reducing action of a small amount of sugar. Accord- ingly it is more satisfactory to use Fehling 's solution for the test. This is made up in two parts, A and B, which are mixed in equal quantities immediately before using. A contains copper sul- phate; B contains sodium hydrate and sodium potassium tar- trate. On mixing these two solutions a deep blue liquid results. The two solutions are kept separate, as otherwise the tartrate will slowly reduce the copper. The advantage in Fehling 's rea- gent lies in the fact that the sodium potassium tartrate unites with the cupric hydroxide to form a complex ion ; thus the cupric hydroxide does not precipitate and does not decompose into the black cupric oxide. On boiling, the liquid remains clear and blue. The combined cupric hydroxide is in equilibrium with a very small amount of this compound in solution so that as fast as the free cupric hydroxide is reduced by the sugar solution, more of the copper-hydrate-tartrate compound dissociates. This complex compound thus furnishes a ready supply of copper hydroxide, and if sufficient sugar is present, all of the copper will be reduced. At this point the blue color will have disap- peared from the liquid. The following equation illustrates the formation of the complex compound:
COO coo
I I
CHOH+2 Cu(0H)2^ CHO— CUOH+2H2O
CHOH CHO— CuOH
I I
COO coo
56 PHYSIOLOGICAL CHEMISTRY
The Fehling test will detect 0.1% glucose in a solution.
Benedict's Test. — The fundamental principle of this test is similar to that of Fehling 's test. In place of sodium hydroxide, sodium carbonate is used, and in place of sodium potassium tar- trate, sodium or potassium citrate. This test has points of superiority over Fehling 's test. In the first place the reagent can be made up in one solutoin instead of two, since the citrate does not tend to reduce the copper, even on long standing. Since sodium carbonate is not so strong an alkali as the hydrox- ide, there is less danger of a trace of sugar being destroyed. Benedict's test will detect a lower concentration of sugar than will Fehling 's, and has replaced it in many laboratories.
Barfoed's Test. — This test also depends upon the reduction of a copper salt (copper acetate) by the sugar. It is performed, however, in acid solution. Barfoed's reagent contains copper acetate and acetic acid. The reducing action of the carbohy- drates is very much less in acid than in alkaline solution. All of the reducing carbohydrates will reduce Barfoed's reagent if boiled a sufficient length of time, producing red cuprous oxide as in the Fehling test. The monosaccharides, however, reduce Barfoed's solution faster than do the disaccharides in equal concentration, so that if properly used, this test may serve to distinguish between monosaccharides and disaccharides. But a concentrated maltose solution will reduce Barfoed's reagent more rapidly than a weak glucose solution, so that this fact should be borne in mind or erroneous conclusions may result.
Almen-Nylander Test. — Nylander's reagent -contains bismuth subnitrate, sodium potassium tartrate and potassium hydroxide. The part played by each constituent is similar to that in Feh- ling's solution. The bismuth subnitrate is reduced to black, metallic bismuth. The equations follow:
Bi ( OH ) ^NOa-f KOH^Bi ( OH ) 3+KNO3
2 Bi(OH)3-fSugar^Bi2+3H2O+(Sugar+30)
Certain substances which interfere with Fehling 's test, have no disturbing influence on the Nylander reaction (uric acid,
CARBOHYDRATES 57
creatinine) so that this test is useful occasionally when the Feh- ling test is of questionable value. A solution containing 0.08% glucose will give a positive Nylander test.
Haines' solution differs from Fehling's solution in contain- ing glycerine in place of sodium potassium tartrate. Its deli- cacy is about equal to that of the Fehling test.
Reduction of Carbohydrates. — By the action of reducing agents carbohydrates may be converted into alcohols, or on fur- ther reduction they may give rise to compounds of the nature of fatty acids. Such transformations apparently occur in the cells of the body, for it is a well known fact that a carbohydrate diet is "fattening." Possibly the carbohydrate molecules are both split into fragments and reduced or dehy- drated, and then recombined to form compounds with longer chains, — fatty acids. The exact mechanism of the process is still unknown. It is quite probable that carbohydrates may give up their oxygen to cells or microorganisms under conditions where vital activities are going on in the absence of atmospheric oxygen. This process is called anaerobic respiration.
Formation of Osazones. — Monosaccharides and many of the disaccharides combine with phenylhydrazine to form osazones. These are yellow compounds which crystallize in needles. The crystals often group together with points at a common center, thus forming rosettes, or fans, or sheaves like grain sheaves. The different osazones have slightly differing crystal forms, but they are best recognized by their melting points; identifying an osazone serves to identify the sugar from which it w^as formed. Glucose and fructose form the same osazone, since the struc- ture of these two sugars differs only around the carbon atoms to which the phenylhydrazine molecules become attached. This test thus will not distinguish between these two sugars. Sac- charose, for a reason to be seen later, does not form an osa- zone, nor do the polysaccharides.
The reaction takes place in three stages as follows:
58 PHYSIOLOGICAL CHEMISTRY
CH2OH CH2OH
I -I
CH OH (CH 0H)3
I I
CH OH -» CH OH
CH OH CH =N NH C^H, +11,0
I CH OH
I CHO +H2N NH C^Hg
The product is a hydrazone. A second molecule of phenylhy- drazine then removes two H atoms from the group next the end carbon atom, and is converted into aniline and ammonia.
|
CH20H 1 |
CH3OH +H,NCeH,+NH3 1 |
|
(CH 0H)3 -» 1 |
(CH0H)3 1 |
|
CHOH +H2N NH CeHg 1 |
c=o 1 |
|
CH=N NH CeHg |
CH=N NH CeH, |
A third molecule of phenyl hydrazine now reacts, forming the osazone.
|
CH^OH (CHOH) 3+ H,N NH CeH. "^ |
CH2OH (CH0H)3 + H,0 1 |
|
Lo 1 |
C=N NH CeHs |
|
CH=N NHCeHg |
C =N NH C0H5 1 |
|
1 H |
In place of phenyl hydrazine, its hydrochloride often is used, as this compound is more soluble and more stable than the free base.
The Molisch Test. — If to a solution containing a carbohy- drate a few drops of 15% alcoholic oc naphthol are added, and
CARBOHYDRATES 59
concentrated sulphuric acid carefully poured down the side of the test tube so that it will form a layer at the bottom of the tube, a violet or reddish ring will form at the juncture of the two liquids. This test will detect not only free carbohydrate, but also carbohydrate in combination with other substances. The reaction is so delicate, however, that it will be given by very small traces of carbohydrate material, even fibers of filter paper (cellulose) so that as a general test it is more valuable in a negative than in a positve result. A negative Molisch reaction is good evidence that carbohydrates are absent, whereas a posi- tive test may be due to the presence of filter paper or other casual impurities. As little as 0.2 mg. filter paper is said to give a strong reaction.
Fermentation — Enzymes
Under the influence of certain microorganisms the carbohy- drates undergo a process known as fermentation, which consists in the breaking up of the carbohydrate molecule to form a variety of simpler compounds. The nature of the products de- pends upon the character of the particular organism respon- sible for the decomposition. Thus the carbohydrate may form carbon dioxide and alcohol, a process known as alcoholic fer- mentation. Or it may form lactic acid, butyric acid, or still other substances. In alcoholic fermentation we may represent the process as follows:
CeH,20e^2 C2H5OH+2CO2
but in reality there are intermediate steps the character of which is but imperfectly understood. The changes brought about in the sugar in these reactions are due to the fact that the microorganisms involved contain or secrete compounds which have the power of breaking down the sugar. We know a great many of these substances, and their activities are by no means limited to the breaking down of sugars. To them has been given the name of enzymes and different members of this class of sub- stances have the power of bringing about the most widely vary-
60 PHYSIOLOGICAL CHEMISTRY
ing chemical reactions, both breaking down substances and building them up.
Enzymes have been the object of much study in recent years, but as yet very little is known of their chemical constitution. There is some evidence indicating that perhaps some of the members of the group may be, or may closely resemble proteins, whereas others appear to be carbohydrates. It is altogether probable that they will be found to vary much in their chemical nature, just as they vary in their chemical activities.
Although little is known of the chemical constitution of the enzymes, many important facts are known concerning their properties and the conditions governing their activities. One of the striking facts of enzyme action is that these substances do not appear combined with the final products which they produce. They are responsible for the breaking down or building up of many classes of compounds, but apparently they only change the speed of reactions which, if given sufficient time, would go on of themselves. Substances which show this behavior are spoken of as catalytic agents, and are said to act by catalysis. Enzymes have been defined as substances produced by living cells, which act by catalysis.
A striking fact in connection with the action of enzjrmes, is that under their influence, reactions go on in the body at a low temperature which, if duplicated in the laboratory without the aid of enzymes, require very high temperatures or the action of ■strong chemical reagents. This is a general property of cata- lytic agent, for we know that in the presence of spongy plat- inum, hydrogen and oxygen will combine at a temperature far below that required in the absence of a catalyst. The final point of equilibrium in the reaction is not altered, — the speed of the reactions is merely altered, and reactions which are capable of being catalyzed are considered to be going on even in the absence of the catalyst, though perhaps at an infinitely slow rate. Catalysts, including enzymes, do not appear com- bined with the final products of the reaction. They are tem- porarily united to the substance acted on, — the substrate, but
CARBOHYDRATES 61
are set free when the action is accomplished. They are thus made available for transforming a new portion of material, and a given weight of enzyme may transform many hundred times its weight of substrate. One might think that under these cir- cumstances the reaction would go on indefinitely, but as a matter of practical experience this is not the case. Various factors contribute to this result. In the first place, there evidently is a gradual destruction of the enzyme. Then also, many reactions of biological importance are reversible, and an accumulation of end products will slow up the process. These products them- selves may affect the speed of the reaction either way. If they tend to speed up the process, the effect is called auto catalysis. If they tend to inhibit the action, the effect is called negative auto catalysis. Then also, there may be a slight amount of per- manent combination between the enzyme and the substrate, thus diminishing the amount of the enzyme. All of these factors described above tend to slow down the reaction except auto- catalysis. Enzymes as a rule behave as do other catalysts, but they are produced only by living cells, are always colloidal which influences their behavior, and are more easily destroyed than are inorganic catalysts. The velocity of enzyme action is that of a unimolecular action (only one substance involved in the reaction).
There are some facts which do not quite harmonize with the classification of enzyme action as truly catalytic. A catalyst is supposed to have no effect upon the character of a reaction, or its final point of equilibrium. The destruction of glucose by enzyme action may yield, however, under the influence of dif- ferent enzymes, widely differing products, — e. g., CO2 and alco- hol, lactic acid, or butyric acid and hydrogen. Apparently the enzymes concerned not only catalyze, but also direct the course of the action.
It was formerly believed that the enzyme of yeast which breal^s down sugar into alcohol and carbon dioxide acted only within the living cell, and that the vital activities of the cell were intimately connected with its action. Organisms of the
62 PHYSIOLOGICAL CHEMISTRY
type of yeast were called ferments. Another class of reactions was known to be independent of the cell by which the active principle was produced; substances responsible for such reac- tions were called unorganized ferments. Among them were the various digestive enzymes. In 1897 this fallacy was cor- rected by Buchner, who ground up yeast cells with sharp sand, thus tearing the cells and allowing the cell fluids to escape. The entire mixture of sand and broken cells was then pressed in a powerful press. A small quantity of liquid was obtained and was filtered through a porous porcelain filter which held back any fragments of cells. The clear liquid was found to decompose sugar in the same way as the original yeast cells had done. The activities of the enzyme were thus in no way dependent upon the vital processes going on in the cell. It is not to be supposed from this that the enzyme activities are of no value to the cell. Quite the contrary is the case, for undoubt- edly the greater number of the chemical reactions which go on in the cell, and which to a large extent make up the sum total of what we call its vital activities, depend upon simple chemical reactions which are directed or controlled by enzymes. We still know certain types of enzymes which thus far have not been isolated from the cells in active form, but possibly this will be accomplished in the course of time.
Enzymes which normally act within the cell by which they are produced are called intracellular, or endoenzymes. Those which normally are secreted, and act outside the parent cell are called extracellular, or exoenzymes. The former are of great interest in pathological processes. The latter include the en- zymes which carry out the processes of digestion in the alimen- tary tract.
Nomenclature and Classification. — The nomenclature of the enzymes is somewhat irregular, as several of the substances were named before any regular plan had been adopted. The ending ''ase" is now employed to indicate that a substance is an enzyme, and the remainder of the word usually indicates either the substance upon which the enzyme acts, the nature of
CARBOHYDRATES 63
the reaction it brings about, or some other property of the com- pound. The substance upon which the enzyme acts is called the substrate. The classification of the enzymes is only pro- visional until a better one is possible. They are grouped accord- ing to the substances acted upon, or the character of the reac- tion induced, thus there are proteases (act on proteins), lipases (act on fats), amylases (act on starch and amylum), lactase (on lactose), maltase (on maltose), oxidases, reductases, etc. Of the older names we have pepsin, rennin, trypsin, zymase (found in yeast), etc.
The suffixes ''lytic" or ''clastic" are frequently used to indi- cate breaking down of the substrate. Many enzyme actions are hydrolytic in character, and the enzymes concerned are classed as sucroclastic, lipoclastic (or lipolytic), proteoclastic (or pro- teolytic) according to the type of substance acted on. Other types are desaminases, which remove the amino group from amino acids, carboxylases, which remove COg from the carboxyl group — COOH, and coagulases, which coagulate proteins.
Specific Nature. — As may be inferred from the above state- ments, the enzymes act only upon particular substances or classes of substances, and in general an enzyme which acts upon one compound will not act upon any other. The enzymes are thus said to be specific in their action, — that is, each one acts only on a particular kind of material, or brings about one parti- cular kind of chemical action. Emil Fischer, one of the most brilliant chemists of all times, has likened this characteristic to the fitting of a key with its lock. As a matter of fact, the en- zymes are believed to fit onto the substances they act upon, form- ing temporary compounds which quickly break down again.
The degree of specificity is truly remarkable, for, of two com- pounds differing only in the slightest detail of stereochemical structure or arrangement of groups, an enzyme frequently will decompose one and leave the other untouched. This strengthens Fischer's key-lock idea. The enzyme is probably taken up, or adsorbed by the substrate and a slight difference in molecular structure may interfere with this process.
64 PHYSIOLOGICAL CHEMISTRY
Influence of Temperature.— One of the characteristics of the enzymes is their extreme sensitiveness to temperature. The temperature of the solution in which an enzyme is acting will be found to influence greatly the speed of the action. The tem- perature at which different enzymes will act best is known as their optimum temperature, and for the enzymes in the body this is in the neighborhood of 37°-40° C, in other words about body temperature. The enzymes in some of the cold-blooded animals, however, whose body temperature varies with that of their surroundings, act well at temperatures much lower than this, and it is obvious that enzymes in plant cells must work well at temperatures much below that of the body. If a solution containing an enzyme is heated to 60° -80° C, the enzyme is de- stroyed, and cooling the solution will not restore its activity. On the other hand, enzymes in general can be exposed to tem- peratures near the freezing point. Their activity is retarded but will return if the solution is warmed.
In the existence of an optimum temperature for each enzyme, these substances differ from inorganic catalysts, for which there usually is no optimum temperature, and which usually are not destroyed by heat, whereas all enzymes are destroyed in solu- tion by heating to temperatures often much below 100° C.
Effect of Chemical Reaction. — Enzymes are also very sensi- tive to chemical reaction. They are destroyed by strong acids or alkalies. The most favorable reaction differs with different enzymes, some acting best in weak acid, others in weak alkaline solution. Certain of the enzymes which act in weak acid solu- tion are destroyed by making the solution even faintly alkaline, and the converse case is also true. Some, on the other hand, will stand considerable variation in this respect, acting in weak acid, in neutral or in weak alkaline solution.
Tenth normal acid and alkali are the limits beyond which enzyme action ceases. The optimum pH for an enzyme is much influenced by a variety of conditions. For example, certain proteolytic enzymes will digest one protein best at one pH, and another protein at another pH. Also the presence of salts or
CARBOHYDRATES 65
other substances may influence the optimum pH for a given action. Langfelt recently has shown that the optimum pH for liver amylase in the presence of chlorides is 6.8, in the presence of phosphates, 6.2 and in the presence of adrenaline, 7.73.
Reversibility. — Some of the enzymes have the power of causing a chemical reaction to go either way, — that is, of de- composing a compound, or under proper conditions of building up the same compound from its decomposition products. Cer- tain of the lipases are notable examples. The property is spoken of as reversibility, and these enzymes are said to be reversible in their action.
This phenomenon was demonstrated by Croft Hill, working with maltose and maltase, and he was able to show that the enzyme caused a resynthesis of disaccharide from the glucose produced. Kastle and Loevenhart on mixing butyric acid and ethyl alcohol with pancreatic extract were able to detect the odor of ethyl butyrate, indicating that the lipase of the pan- creas had caused a recombination of these substances to form the ester. The importance for the cell of the reversibility of enzyme action is apparent, for much of the work of the cell must be synthetic, either in building up or repairing its own structures, or in producing reserve materials for storage, such as glycogen, fats, lipins, or other substances.
Active and Inactive Form. — In the form in which they are secreted by cells, some of the enzymes are inactive, and become capable of exerting their customary activity only after they have been acted on by some other substance. The enzymes are thus said to exist in inactive and active forms.
The inactive form is often called the zymogen form; the activating substance, the coenzyme or kinase. The coenzyme may be a salt, such as a chloride or phosphate, an acid, for example the gastric hydrochloric acid which activates pepsino- gen. Coenzymes are usually unaffected by heat, so they obvi- ously are not themselves enzymes. They often dialyze, and many enzymes are rendered inactive by dialyzing off the salts or other dialyzable substances in the solution.
66 PHYSIOLOGICAL CHEMISTRY
Action Retarded by Products.— The activity of an enzyme is often retarded by the products which it produces from the sub- strate. In life (in vivo) these products usually are removed, as in absorption of the digestion products from the intestine. In a laboratory experiment in a test tube or beaker, this may influ- ence the extent of digestion by an enzyme to a considerable ex- tent. This fact may be summed up by saying that enzyme ac- tion often is incomplete in vitro (in glass).
This topic already has been discussed under ''autocatalysis" at an earlier point in the consideration of enzymes.
Progressive Action. — The enzymes are often said to be pro- gressive in their action. This is only a special case of their specific nature. Many reactions depend upon more than one enzyme for completion, the reaction taking place in successive stages, each one of which is brought about by a different enzyme. Thus the breakdown of starch into glucose requires at least two and possibly more enzymes. The first breaks down the starch through various stages to maltose, the second breaks the maltose into glucose.
Antienzymes — Defensive Enzjmies
If a rennin solution is injected into the blood of an animal, a substance appears in the blood which is capable of destroying the rennin. Such a substance is called an antienzyme or para- lyzer. If various other substances, foreign to the body, are introduced into the blood, enzymes are produced capable of destroying them. Abderhalden has called these substances ' ' de- fensive enzymes." This is referred to later in the discussion of the Abderhalden reaction for pregnancy. It truly is remark- able that the body tissues should be able to produce such sub- stances for defense against compounds which they surely never can have been called upon to meet, either in the development of the individual or of the race. The secret to this problem may hold the explanation of the mode of enzyme production and enzyme action.
Summary. — In summary it may be said that enzymes are sub-
CARBOHYDRATES 67
stances of unknown chemical constitution, which act as catalytic agents affecting a large variety of chemical reactions. They are specific in their action, they are sensitive to changes in reaction and temperature, being destroyed if heated to 60° -80° C. Some are reversible in their action, they are often secreted in inactive form and become active only on coming in contact with some other substance, their action is often incomplete in vitro, and frequently is progressive in its character.
On the activities of enzymes depend a very large number of the processes by which the cells, and hence the tissues and the body as a whole carry on their various activities.
Individual Groups of Carbohydrates.
Pentoses. — The pentoses are found in both plants and animals, usually combined with other substances as in nucleo- proteins or in the form of polysaccharides made up of many molecules of pentose. They are obtained by the hydrolysis of these compounds. A pentose, probably arabinose, has been found in the urine. This condition is known as pentosuria. Pentoses may be either aldoses or ketoses. They reduce Fehling's and other similar solutions, and give osazones with phenylhydrazine. They usually do not ferment, however. The pentoses are utilized by herbivorous animals but the extent to which they may be utilized by man seems to be more limited, although the subject is still a matter of some uncertainty. Pentoses may be distinguished from hexoses by their osazones, by their failure to ferment readily, and also by certain color reactions among which are the orcin and phloroglucin tests. If a pentose is heated with concentrated hydrochloric acid and a little orcin or phloroglucin, a distinct color change results. With orcin the color is first vio- let, then blue, red, and finally green, and a bluish green pre- cipitate forms. With phloroglucin, the color is red. As other substances will give similar colors, it is necessary to confirm the result by observing the absorption spectrum of the colored sub- stance after it has been dissolved out by shaking the liquid with amyl alcohol. The orcin test gives an absorption band between
68 PHYSIOLOGICAL CHEMISTRY
the C and D lines, the phloroglucin test, between the D and E lines. See discussion of absorption spectra below.
This procedure does not distinguish pentoses from glucuronic acid, however.
Arabinose is obtained by the hydrolysis of gum arabic, cherry gum, peach gum, etc., with dilute acid. It sometimes occurs in the urine. It has a sweet taste, and its solution is dextrorotatory (1-arabinose has a specific rotation + 104.5°). Its melting point is 160°. Its osazone melts at 163°-164°. Xylose is obtained by hydrolyzing wood, gum, straw, bran, etc. It often is called wood sugar. The pentose isolated from the nucleoprotein of the pan- creas is said to be d-ribose. Its solution is levorotatory. The specific rotation of 1-xylose is + 18.1°. Its phenylosazone melts at 155°-158° C.
Absorption Spectra.
White light is made up of a great many different colored lights as may be demonstrated by passing a beam of white light through a prism and observing the spectrum resulting from spreading out the different colored components of the original ray. Colored light may be either light of a single color or wave length, or it may be a mixture of many different colored lights, the sum of which, however, falls short of making a complete spectrum, or in which one color greatly predominates in in- tensity over the other colors present. If light passes through water, it still appears as Avhite light, for the water has allowed the ray to pass through intact. If a ray of white light passes through a solution of hemoglobin, the red pigment of the blood, or of an amyl alcohol solution of the compound made by heating a pentose with orcin and hydrochloric acid, the ray no longer looks white, because the solution has absorbed or reflected a por- tion of the colored light, allowing only the red and perhaps some neighboring kinds of light to pass through. If the light is spread out in a spectrum, a portion will be missing. Many substances in solution have the property of absorbing particular kinds of light in this way, thus leaving a blank dark area in
CARBOHYDRATES
69
the spectrum if the emerging light is analyzed by spreading it out into its spectrum. The property is so constant that it may be made use of to identify compounds. The spectra resulting are called absorption spectra, and each absorption spectrum is characteristic of a particular substance. The lines or dark areas are charted with reference to the dark lines always ob- served in the spectrum of sunlight. These lines are called the Frauenhofer lines. The important ones are as follows:
C D
Red Yellow
Eb F
Green Purple
By looking through a solution with a spectroscope it thus is possible to identify many compounds of biological importance.
Hexoses. OqH^zOg
Glucose. (Dextrose, grape sugar) . — Glucose is found in both plants and animals. In the plant world it occurs in grapes and other sweet fruits, in seeds, roots, etc., and as a constituent of di- and polysaccharides and glucosides is much more widely distributed. In animals it is found in the blood and lymph, and occasionally in the urine. If the amount in the urine is more than a trace and it is found regularly, the condition is pathologi- cal and no time should be lost in consulting a physician. Glu- cose also is found in honey, an animal product. It may be ob- tained by boiling starch, glycogen, dextrins, etc., with dilute acid. Glucose crystallizes readily. It is soluble in water. The solution is less sweet than that of cane sugar. It is dextrorota- tory, the specific rotation varying somewhat with the concen- tration. The figure usually reported is +52.5° for a solution in which equilibrium has been reached. It shows strong mutarota- tion. It is slightly soluble in warmi alcohol and insoluble in
70 PHYSIOLOGICAL CHEMISTRY
ether. It gives all the reduction tests, ferments readily with yeast, forms caramel on warming with alkali, and with phenyl- hydrazine forms an osazone which melts at 205°. It is perhaps the most interesting of the sugars, for it is found in the blood, and serves as one of the most valuable fuels for the body cells. By the oxidation, or burning of glucose the cells produce heat and do mechanical work.
Fructose. (Levulose, Fruit Sugar.)— Fructose is found in plants chiefly combined with glucose as cane sugar, or in the polysaccharide inulin from which it may be obtained by hydro- lysis. It also occurs in honey. It is sometimes, though rarely, found in the urine in a condition known as levulosuria. The solu- bilities of fructose are similar to those of glucose. Its solution rotates the plane of polarized light to the left, the specific rota- tion being — 92°. It is called d-fructose because of its struc- tural relationship to d-glucose, so that in this case the ''d" does not indicate that the compound is dextrorotatory. Solutions of fructose show the phenomenon of mutarotation. Fructose is a ketone sugar, and gives all the usual reduction tests for car- bohydrates. It forms the same osazone as glucose, so that this test is of no value to distinguish the two sugars. Fructose also ferments with ordinary yeast. Fructose forms a calcium com- pound which is much less soluble than that of glucose and serves to separate the two sugars when they occur in a mixture.
Fructose may be distinguished from glucose by its levorota- tion, and also by the Seliwanoff reaction. On adding a few crystals of resorcinol, and concentrated hydrochloric acid to a levulose solution, and heating, a red color results. Glucose will give the test under certain circumstances, however, so that it must be carried out under definite conditions or it may lead to erroneous conclusions.
It also is possible to distinguish these sugars by means of methylphenylhydrazine. With this substance, fructose and other ketoses form osazones, whereas glucose and other aldoses form only hydrazones.
d-Galactose. — Galactose occurs in nature as a constituent of several gums, in the polysaccharide galactan in sea-weed, as a
CARBOHYDRATES 71
constituent of lactose or milk sugar and in certain substances in brain and nerve tissue. It is prepared from milk sugar or from various gums by hydrolysis. Galactose is somewhat less soluble in water than glucose. The solution is dextrorotatory, the spe- cific rotation being +81°. Galactose is an aldose and gives the usual reduction tests, and forms with phenylhydrazine an osa- zone which melts at 192°-195°. Galactose ferments slowly but completely with ordinary yeast. If heated with nitric acid it forms mucic acid which is relatively insoluble and forms a fine white precipitate. This test serves to distinguish galactose from all sugars except lactose. It is of interest that the mammary gland constructs it out of the glucose of the blood, and unites it with glucose to form lactose or milk sugar.
Amino Sugars. — Closely allied to the monosaccharides are the amino sugars, which differ from the simple sugars only in having an amino group ( — NHg) instead of an — OH attached to the carbon atom next the aldehyde group. These compounds have been obtained by the hydrolysis of complicated substances occurring in the shells of lobsters and from the proteins mucin and mucoid which are widely distributed in the animal world. d-Glucosamine is an important member of this group. It is ob- tained by boiling the chitin of lobster shells with hydrochloric acid. It is readily soluble in water, the solution being alkaline. Its hydrochloric acid salt shows solubilities similar to those of the monosaccharides. The solution is dextrorotatory, having a specific rotation of from + 70° to + 74° according to concen- tration. It reduces the ordinary carbohydrate reagents and gives an osazone identical with that formed from glucose. Gluco- samine does not ferment, however. d-Glucosamine is an inter- esting compound because in composition it stands midway be- tween the carbohydrates and a group of substances called amino acids, which are the simple units of which the proteins are com- posed.
d-Glucuronic Acid. — This compound is obtained from glucose by oxidation, and is found in the body in combination with other substances. These compounds are called conjugated glu- curonates. Glucuronic acid has the formula:
72 PHYSIOLOGICAL CHEMISTRY
COOH
I
H— C— OH
I H— C— OH
I HO— C— H
I H— C— OH
I CHO
Glucuronic acid
It is interesting chiefly because it combines with various pro- ducts formed by putrefaction in the intestine such as phenol, indol, skatol, etc. These substances are absorbed into the blood stream, and would exert a toxic influence upon the cells if it were not for the fact that the body promptly unites them to glucuronic acid, (or some other compounds) forming the con- jugated glucuronates which are relatively harmless, and are excreted in the urine. This is one of nature's protective de- vices for shielding the cells against the influence of injurious substances. Solutions of glucuronic acid give the same reduc- tion tests as glucose, are dextrorotatory, but do not ferment. The conjugated glucuronates are strongly levorotatory.
Disaccharides.
The disaccharides are formed by the union of two molecules of monosaccharide with loss of water.
C^cHisOg + O6H12O6 —> C12H22OH + H2O.
By the action of dilute acids, enzymes, etc., they may be split into their constituent monosaccharides. The three disac- charides of importance are saccharose, lactose and maltose. These sugars are of great importance as food substances.
Saccharose. (Sucrose, Cane Sugar.) — Cane sugar is found in many plants, notably in the juice of the sugar cane, which contains about 20%, and in carrots. It is found in many sweet fruits, such as the banana, strawberry, pineapple, etc., and in
{
CARBOHYDRATES
73
the sap of the sugar maple. It is a valuable food substance, and serves also as a condiment, by its sweetness making other foods more palatable. Cane sugar is prepared by treating the sap or juice containing it with milk of lime. This neutralizes any acids present, which otherwise would hydrolyze the sugar during evaporation. After being boiled to remove the protein, the calcium is removed by running in carbon dioxide, and the solution is decolorized either with animal charcoal or sulphur dioxide. After being boiled and filtered the liquid is evaporated in vacuo, and the cane sugar crystallizes out. The remaining liquid is known as molasses, and still contains considerable quan- tities of sugar which may be obtained by precipitation as cal- cium or strontium saccharate. From this compound the cane sugar may be set free with carbon dioxide.
Cane sugar is readily soluble in water, less so in alcohol, and insoluble in ether. The aqueous solution is very sweet, and is strongly dextrorotatory, the specific rotation being + 66.5°. The specific rotation of saccharose is practically independent of changes in concentration and temperature, so that the property is often made use of for its estimation. On hydrolysis it yields glucose and fructose.
On being heated to about 160° cane sugar melts and if al- lowed to cool, forms a glassy mass which is known as barley sugar. At about 200° it turns brown, forming caramel.
CH^OH
CHOH
I
CH
CHOH ! I 0
CHOH I
I 1
CH
O
CH.OH
CH.OH
Saccharose
74 PHYSIOLOGICAL CHEMISTRY
Cane sugar does not reduce the usual carbohydrate reagents, such as Fehling's or Benedict's solutions and it does not form an osazone if treated with phenylhydrazine. This is due to its mo- lecular structure. The aldehyde and ketone groups of its con- stituents are no longer free. On long boiling with these reagents, however, or after the action of an inverting enzyme on the cane sugar, the solution will give positive reactions with the above re- agents since the sugar is split into its constituent parts. The hy- drolysis or splitting of cane sugar is called inversion because the solution, originally dextrorotatory, is levorotatory after hydroly- sis. This is due, of course, to the fact that the hydrolyzed solu- tion contains equal quantities of glucose and fructose, the latter of which rotates more strongly to the left than does glucose to the right. The mixture is known as '^invert sugar." A solu- tion of cane sugar ferments readily with ordinary yeast, which contains an enzyme invertase which will invert the cane sugar. The resulting glucose and fructose are fermented by the zymase.
Lactose. — Lactose is found in the milk of all mammals, but does not occur in plants. Cow's milk contains about 4% lac- tose, human milk about 5% to 7%. It may occur in the urine of women during pregnancy. Lactose is prepared from whey. On concentration, lactose crystallizes out, and may be purified
|
CH^OH |
H C = 0 1 |
|
CHOH 1 |
CHOH 1 |
|
CH |
CHOH 1 |
|
CHOH ( 1 |
3 CHOH |
|
CHOH 1 |
CHOH 1 |
|
CH— 0 |
CH„ |
|
ralactose |
Glucose |
Lactose
CARBOHYDRATES 75
by recrystallization from water. The crystals are hard and gritty, and the solution is not so sweet as that of cane sugar. Lactose is composed of one molecule each of glucose and galac- tose. It is manufactured in -the breast gland from the glucose of the blood. This is an interesting example of the conversion of one sugar into another in the body, since a portion of the glucose must be changed into galactose.
Lactose responds readily to the reduction tests for the mono- saccharides. It thus possesses a free aldehyde group, and is represented by the accompanying formula. The solution of lactose is dextrorotatory. The specific rotation is + 52.5°. Lac- tose does not ferment with ordinary yeast. This property is serviceable in identifying it. It should be remembered that lac- tose will give the mucic acid test, since it contains galactose.
Maltose. — Maltose is formed in the hydrolysis of starch or glycogen by amylase. Since this enzyme is widely distributed in both plants and animals, maltose may be found wherever there is starch or glycogen.
Maltose is readily soluble in water. The solution is not so sweet as that of cane sugar. The solution is dextrorotatory, the specific rotation being -|-136°. This value varies, however, with concentration and temperature. Maltose reduces Fehling's re- agent, etc., and gives an osazone. Its structure is thus con- sidered to be similar to that of lactose which has one free al- dehyde group. On hydrolysis it yields two molecules of glu- cose. Maltose ferments readily with yeast. It may easily be dis- tinguished from glucose by hydrolizing with dilute acid. After hydrolysis the reducing power of the solution will be found to have altered, since two molecules of glucose are now present for every molecule of maltose destroyed.
Polysaccharides.
Members of the polysaccharide group differ from one another considerably in their solubilities and other properties. They are found in both plants and animals in which they form reserve supplies of food material, and in plants and some of the lower
76 PHYSIOLOGICAL CHEMISTRY
animals, they are important constituents of the supporting framework or protective covering. The members of this group are made up of several molecules of monosaccharide united with loss of water to form the larger polysaccharide molecule. The commoner individual polysaccharides yield only one kind of monosaccharide when hydrolized, thus differing from the other material bases which, on hydrolysis yield varying kinds of sim- pler units. Since the number of monosaccharide molecules which, make up a polysaccharide molecule is unknown, it is customary to express the formula with the indefinite coefficient n.
Starch (CeHjoOg)!!. — Starch is a plant product, and is found stored in leaves, seeds, fruits, tubers, etc. Grains contain as much as 50-70% of their dry weight; potatoes contain from 15-30% of their wet weight. Starch forms granules of more or less characteristic shapes which are serviceable in determining the source of the starch. Thus potato starch appears as egg- shaped granules which often show concentric lines. Starch is prepared from potatoes or grain by grinding the material, filter- ing through sieves to remove the coarse debris and allowing the suspended starch particles to settle. It forms a white amorphous powder which does not dissolve in cold water. If boiled with water, the granules are broken open and the starch forms an opalescent solution. Starch is insoluble in alcohol and ether. A starch solution rotates the plane of polarized light to the right. It will not reduce Fehling's, or similar solutions, and will not ferment. The characteristic starch test is the formation of a blue color on the addition of a few drops of iodine solu- tion. This color disappears on heating, but reappears if the solution is cooled. Alkali and alcohol also destroy the blue color. On boiling with dilute acids starch is broken down to glucose. The starch passes through various intermediate stages, the nature of which may be followed by the iodine test. If portions of the hydrolizing mixture are tested with iodine from time to time, the blue color soon gives place to red. This cor- responds to the stage known as erythrodextrin (from the Greek word meaning ''red"). On further hydrolysis, the iodine test
CARBOHYDRATES 77
gives no color. This is the achroodextrin stage (Greek word means *'no color"). Beyond this stage maltose is formed, which then breaks up into glucose. The appearance of reducing sugars may be recognized, since the mixture will reduce Feh- ling's solution.
Starch is an enormously important food substance, and is widely used in the arts. In stiffening linen, the starch is broken down into dextrins by the heat of the iron. These dextrins give the fabric its stiffness and glossy appearance.
Dextrins. — Little need be said of the dextrins in addition to the fact that they are intermediate stages formed in the hydrol- ysis of starch to glucose. There probably are many members of the group, but so far, little is known of the different individ- ual dextrins. Dextrins themselves probably do not reduce Feh- ling's solution, or only slightly, but commercial dextrin, which is prepared by the partial hydrolysis of starch, usually reduces Fehling's solution slightly, probably because the mixture con- tains maltose or glucose as the result of complete hydrolysis of some of the material. Dextrin solutions do not ferment, and give a red color (erythrodextrin) or no color (achroodextrin) with iodine according to the extent of the hydrolysis. Dex- trins are readily soluble in water, but are precipitated by the addition of alcohol. The aqueous solution is dextrorotatory.
Inulin. — Inulin occurs in the sap of various plants and is found to the extent of 10-12% in the tubers of the dahlia. It is soluble in hot water, and gives a yellow or brownish color with iodine. It is interesting chiefly because on hydrolysis it yields levulose instead of glucose. It does not reduce Fehling's solution, and its solution is levorotatory.
Gums and Mucilages. — The gums and mucilages are widely distributed and on hydrolysis yield various pentoses and hex- oses, and other substances. They vary greatly in their solubil- ities.
Cellulose. — Cellulose is chiefly important in forming a large part of the structural framework of plants. The plant cell walls are made up of cellulose mixed with lignin and other sub-
78 PHYSIOLOGICAL CHEMISTRY
stances. Cellulose also is found in certain lower animals, the tunicates. Cellulose is insoluble in the ordinary solvents. It dissolves, however, in Schweizer's reagent, an ammoniacal solu- tion of copper oxide, and in some other reagents. Cellulose derivatives are extensively used in the arts. Thus nitroeellu- loses of varying composition are used in the manufacture of ex- plosives, collodion, celluloid, artificial rubber, etc. From cellu- lose artificial silk and artificial gutta percha also are prepared.
There has been much discussion as to whether cellulose is of value as a food. Undoubtedly this is the case in herbivora. In man it probably is of little food value, but serves a useful pur- pose in giving bulk to the food and thus stimulating the mus- cular activity of the intestine. It has been stated that the cellu- lose of young and tender lettuce, asparagus, etc., may be utilized by the body to a considerable extent as food. There is no enzyme in the digestive juices capable of hydrolyzing cellulose, so that its disintegration must be due to the action of intestinal bac- teria.
Glycogen. — Glycogen is found in the organs and tissues of animals, and also in some plants (yeast). It serves as a reserve fuel or food supply. The chief depots for glycogen deposit are the liver and the muscles. Glycogen is found in oysters, scallops and other molluscs. Glycogen may be prepared from the liver of an animal which has just been killed. The liver is ground in a mortar with sand, and extracted with boiling water slightly acidified with acetic acid. If the extraction is not made at once after the death of the animal the glycogen supply will be greatly diminished or disappear altogether, as it is rapidly hydrolized to glucose by autolytic enzymes in the Liver tissue. Feeding rabbits for a day or two on carrots before killing will insure a liberal supply of glycogen in the liver.
Glycogen is a white amorphous powder, which dissolves in cold water forming an opalescent solution. This solution gives a wine red or brown color with iodine. It does not reduce Feh- ling's solution, is not fermented by yeast, and is dextrorotatory. On boiling with dilute acids, glycogen is hydrolyzed to glucose.
CARBOHYDRATES 79
After hydrolysis the solution will of course, reduce Fehling's solution.
Glucosides.
Many compounds are obtained from plants and animals which yield on hydrolysis varying quantities of glucose or other sim- ple sugars and in addition a wide variety of other compounds. To this group is given the name glucosides. Among these sub- stances are many of the drugs used in medicine. The di- and polysaccharides themselves may be looked upon as glucosides, since in them glucose is combined with one or more sugar molecules.
CHAPTER IV FATS, PHOSPHATIDS AND ALLIED SUBSTANCES
Distribution and Importance. — The fats are widely distrib- uted in nature, in both plants and animals. In the former they are found in seeds such as cotton seed, the castor bean, etc., in fruits, such as olives, in nuts and also in the leaves and roots of some plants. In animals they are found in most tissues and fluids. The amounts in the tissues vary considerably. The ac- tive living protoplasm contains only about 1-10%, whereas mar- row, fatty tissue, etc., may contain considerably over 90%. The fats are of importance as fuels for the body. They are laid away in large deposits which also serve the purpose of insulat- ing the body by forming a blanket layer which aids in the con- servation of heat. There is a layer of subcutaneous fat, and there are also large deposits around the abdominal viscera. Considerable quantities are found in the intramuscular con- nective tissue.
Composition and Structure. — The fats are made up of car- bon, hydrogen and oxygen. The oxygen is present in much smaller per cent than in the carbohydrates. The constituent parts of the fats are the triatomic alcohol glycerine or occa- sionally some other alcohol, and organic acids, either of the fatty acid or a similar series. It is of interest that the acids making up the body fats have even numbers of carbon atoms in their molecules. The following list gives the names and formulas of some of the important acids:
Butyric CH.CH^CH.COOH (C^H^OJ
Caproic CH3CH2CH2CH2CH2COOH (CeHi^OJ
Caprylic CH3(CH2)eC00H {CJi,,0,)
Capric CH,(CH2)8C00H (C^.H^oOJ
Palmitic CH3 ( CHo ) i^COOH ( Ci.Hs^O^ )
Stearic CH3(CH2)ieCOOH (CigHgeOJ
80
FATS, PHOSPHATIDS, ETC. 81
The acids listed above are all saturated compounds, that is they contain no double bonds. An acid found in a large number of fats is oleic acid, which has the same number of carbon atoms as stearic acid, but two less hydrogen atoms. It is thus CigHg^Og and its formula is
CHaCCHJ.CH = CHCCHJ.COOH.
It is an unsaturated acid, and contains a double bond. Fats containing this acid have a lower melting point than those con- taining the corresponding saturated compound. Some allied compounds contain other alcohols in place of glycerine. Thus cetyl alcohol C1CH3.5OH is found in spermaceti in the head of the sperm whale, and myricil alcohol CsoHg^OH in beeswax, etc. Esters of these alcohols usually are called waxes. The follow- ing formula illustrates the structure of a fat.
0
II CH2O— C— K
0
II CH— 0~C— R
0
II CH2— 0— C— R
R is the rest of an acid molecule. If the fat were tristearin, R would represent a chain of 16 CHg groups with a CH3 group at the far end. Fats are thus tri-atomic esters of glycerine and an organic acid. The three R's may be all the same fatty acid, or they may be different. There is thus the possibility of having a large number of different fats, differing in the kind of fatty acid present. This possibility is realized in nature, and a large number of different fats are known. The naturally occurring fats are rarely made up of a single kind of fat, but usually are mixtures of various kinds such as tripalmitin, tristearin, and triolein, as the fats from these respective acids are called. Oleic acid has a very low melting point, and triolein also melts at a low temperature. The presence of much triolein in a fat lowers
82 PHYSIOLOGICAL CHEMISTRY
its melting point, often to such an extent that the fat is liquid at ordinary room temperature. Such fats are called oils. Other unsaturated acids are found in some fats, or ''oils," and they exert a similar influence.
Among these unsaturated acids are linolic (CigHggOs) and linolenic (CigHgoOJ. Oils which contain considerable amounts of the esters of these acids undergo oxidation easily and are converted into a hard resinous material when exposed to air and light. They are called drying oils, and are used in paints and varnishes. Linseed oil is an example of this group. Others, such as cottonseed oil only thicken on exposure to air and light; they are called semidrying oils. Olive oil does not harden under similar circumstances and is classed as a nondry- ing oil.
General Properties.— The solid fats are white or light yellow substances, which if pure are odorless and tasteless. The oils often are yellow and frequently have a decided taste and odor. They are insoluble in water, somewhat soluble in cold alcohol but much more so in hot alcohol, and soluble in ether, chloro- form, benzol, etc. From solutions, the fats often may be ob- tained in crystalline form as long needles. The specific grav- ities of all the fats and oils are less than that of water, hence they float at the surface. They reduce the surface tension of water. The naturally occurring fats and oils do not have sharp melting points, since they are mixtures of different kinds of fats. Even pure fats often show much indefiniteness in melting point. Some melt, resolidify at a slightly higher temperature, and if further warmed, melt again. This is supposed to be due to the fact that an internal rearrangement in the molecule is brought about by heating.
Emulsification. — If neutral oil and water are shaken to- gether vigorously, and the mixture allowed to stand, it will quickly separate into two layers, oil and water. If a small amount of soap solution is added to this mixture and the shak- ing repeated, the liquid becomes milky in appearance, and even after prolonged standing will fail to separate into two layers.
FATS, PHOSPHATIDS, ETC. 83
The fat is said to be emulsified. On examining such a mix- ture under the microscope, it will be seen to be filled with minute globules of oil suspended in the water. If the propor- tions are reversed, that is, if there is much oil and little water, the water will be suspended in the oil. Soap is by no means the only substance which will favor the formation of an emul- sion. Albumin, gums such as gum arabic, and a variety of other compounds will bring about a similar result. Lymph will emulsify a fat, and if a drop of lymph and a drop of oil are brought in contact on a microscope slide, the oil may be seen to break up into minute droplets and enter the lymph drop. Physiologically the formation of emulsions is of great im- portance. In digestion in the stomach only emulsified fats are attacked to any extent. In the intestine, fats of the food are emulsified by the pancreatic juice, a process which is extremely important for their proper digestion and absorption. The mechanism of emulsion formation has been the subject of much study. Probably different emulsifying agents act in different ways, or a single substance may act in more than one way. It is believed that the soap, albumin, etc., collects around the tiny fat droplets and serves to insulate them and thus lessen the tendency to run together. The lowering of the surface tension is also a factor in the production of emulsions, and possibly also electrical forces tending to repel the similarly charged particles of fat. Milk is an example of a fairly perma- nent emulsion. The fat droplets are suspended in a liquid which contains protein. On standing, a considerable portion of the milk fat finally will float to the surface. When removed from the skimmed milk beneath, this is known as cream. On churning, the emulsified fat runs together and butter is formed.
Saponification. — If a fat is boiled with an alkali, or an acid, it is split into fatty acids and glycerine. This process is known as saponification. If an alkali is used, the fatty acids react with the alkali to form salts. These salts of the higher fatty acids have a slippery feeling, and their solutions foam on being
84 PHYSIOLOGICAL CHEMISTRY
shaken. They are called soaps. The accompanying equation illustrates the process :
0
II CH^O — C. C,,H35 CH^OH
0
CHO — C . C17H35 3 NaOH -^ CHOH + SCi^Hg^COONa
I O + I
I II I
CH2O — C . C17H3, CH2OH
The process is best carried out in alcoholic solution. Sodium soaps are known as hard soaps; potassium soaps which are but- tery in consistency are known as soft soaps. Calcium soaps are very hard and insoluble. Soap is a useful cleansing agent. Soiled articles, — clothing, the hands, etc., usually are covered with a layer of fatty material which entangles and holds parti- cles of insoluble inorganic dirt. Soap emulsifies the fat and the remaining material is carried away by the water or by the lather, which takes up the particles of dirt mechanically.
Since calcium soaps are very insoluble, hard water is not good for washing purposes, as the calcium precipitates the soap added, and thus interferes with its cleansing activities.
Rancid Fats. — Many natural fats, upon standing, acquire a disagreeable taste and odor. This is due to the splitting of some of the neutral fat into glycerine and fatty acids. The lower fatty acids, such as those found in butter have a very disagreeable taste and odor, hence the character of ''rancid" butter, etc.
Detection and Identification. — Acrolein Test, — Fats are easily detected by their physical properties, such as solubility, appear- ance, greasiness, etc. A test given by all common fats is known as the acrolein test. If a fat is heated to 300° it is de- composed. The glycerine portion of the molecule loses water and forms the unsaturated compound acrolein. The test is obtained more readily if the fat is heated with a dehydrating
FATS, PHOSPHATIDS, ETC. 85
agent such as potassium acid sulphate, boric acid or phosphorus pentachloride. Acrolein is easily recognized by its extremely sharp and irritating odor. Since only substances containing glyc- erine give the test, it may be used to distinguish between fats and fatty acids or soaps.
|
CH20H CHOH 1 |
— 2H20-> |
CH2 II CH 1 |
|
CH2OH |
CHO Acrolein. |
Melting Point. — The melting points of the natural fats are not sharp, since natural fats usually are mixtures. They often melt, solidify on further heating, and melt again at a higher temperature. Those fats whose melting points are below or- dinary room temperature are called oils. The melting points of fats in animal tissues are generally below the usual tempera- ture of those tissues, so that the body fats are in a fluid state. The fats of cold blooded animals melt at lower temperatures than those of warm blooded animals.
Saponification Equivalent. — The saponification equivalent is the number of milligrams of potassium hydrate necessary to neutralize the fatty acids produced by the saponification of one gram of fat. The smaller the molecular weight of the acids in the fat, the larger will be the number of molecules in a gram, and the higher the saponification number. Fats made up of fatty acids such as palmitic, stearic and oleic acid such as oleo- margarine have a saponification number around 195. Butter, which contains fatty acids of low molecular weight has a saponification number around 227. These two substances may be distinguished easily in this way.
Volatile Fatty Acids. — Reichert-Meissl Numher. — This method is used to give evidence of the amount of lower fatty acids in a fat. The fat is hydrolized with alkali, acidified with sul- phuric acid and distilled. The fatty acids of low molecular weight distil over and may be titrated. Those of higher molec-
86 PHYSIOLOGICAL CHEMISTRY
ular weight are not volatile and remain behind. The number of cubic centimeters of N/10 alkali required to neutralize the volatile fatty acids from 5 grams of fat is called the Reichert- Meissl number. For butter fat the number is 25-30.
Iodine Number. — The acids which contain double bonds, such as oleic acid will take up iodine or bromine, adding on two atoms of the halogen for each double bond. By this process it is possible to determine how much unsaturated acid is present in a fat. The weight of iodine in centigrams taken up by a gram of oil is known as the iodine number. Hydrogen and oxygen are taken up in an analogous manner, and this fact is also used to identify fats.
Acetyl Eqivalent. — Some fats contain oxyacids, — that is acids containing hydroxyl groups. These groups may be re- placed by acetyl groups, which in turn may be split off and the resulting acetic acid titrated. The number of milligrams of potassium hydrate required to neutralize the acetic acid ob- tained from 1 gram of fat in which the hydroxyl groups have been replaced by acetyl groups is known as the acetyl equiva- lent.
The various factors described above have been determined for the important natural fats, and variations in one or more of these characteristic constants are of great service in deter- mining whether a given fat or oil is pure, or has been adul- terated.
Important Fats. — Tristearin, tripalmitin and triolein are the three fats occurring most frequently in natural fats.
Tristearin, or stearin, as it is often called, melts first at about 55°, resolidifies and melts again at about 71°. It is a hard, flaky material, and the least soluble of the three. It is obtained from tallow. Mixed with a little paraffin to make it less brittle it is moulded into candles. Free stearic acid is found in old pus, in gangrenous or tuberculous masses, etc., where decomposition of fat has taken place. It is found as its alkali soap in blood, bile, etc., and as its calcium soap in the feces.
Tripalmitin, or palmitin, is found in all animal and most
FATS, PHOSPHATIDS, ETC. 87
vegetable fats, notably in palm oil, whence it derives its name. It predominates in human fat. It melts first at about 50°, re- solidifies, and melts again at about 66°.
Triolein, or olein, is found in animal, and to a greater extent in vegetable fats and oils. It melts at — 6° C, and is thus a liquid (an oil) at room temperature. The presence of olein lowers the melting point of natural fats. If exposed to the air olein quickly becomes rancid. Oleic acid, by reason of its double bond will take up iodine as described above.
Butter. — Butter, the fat obtained from cream, contains about 85% fats. About 7% of this is made of lower fatty acids. Of the remainder 60-70% is palmitin, and 30-40% olein. Butter contains very little stearin.
Oleomargarine is made usually from beef fat. The fat is melted, cooled, the oily portion pressed out and this mixed with various substances such as peanut oil, lard, etc., and finally churned up with milk to give it a butter flavor. If oleomarga- rine is made from clean, wholesome materials it is a perfectly sat- isfactory food substance. It often lacks certain substances found in butter, however, which are called vitamines, which recently have been shown to be very valuable materials to the body. Cod liver oil also contains these substances. Perhaps this fact justifies the popular impression of its beneficial nature.
Lanolin, or yjool fat, which is an ester of higher fatty acids chiefly with the alcohol cholesterol in place of glycerine, is use- ful in medicine. It forms extremely fine emulsions and thus serves as a valuable basis for the formation of salves and oint- ments. Substances containing alcohols other than glycerine, such as cholesterol are often classed as waxes.
Phosphatids, Cerebrosides, and Sterols
A large number of substances have been isolated from animal and plant tissues which have certain similarities to the fats, such as solubilities, general appearance, etc. Some of these compounds are also related chemically to the fats. Substances of this nature are found in all cells, both
88 PHYSIOLOGICAL CHEMISTRY
animal and vegetable, and particularly in the brain and nerve tissues. Little is known of the biological function of these com- pounds, although as our information increases, they appear to be increasingly important. The composition of some of them is known, whereas in the case of others, we have no assurance that the substances reported are single compounds and not mix- tures of closely related compounds.
The term lipoid has been used to designate these ''fat-like'' bodies, but the term has been used in so many different con- nections that MacLean suggests it be abandoned. He proposes the name ''lipins" for the two groups, phosphatids and cere- brosides. These substances, which may be obtained from various tissues by extraction with ether, yield, among other things, fatty acids on hydrolysis. The phosphatids contain both nitro- gen and phosphorus, and on hydrolysis yield phosphoric acid, glycerine, fatty acids, and a nitrogen base such as choline or amino-ethyl alcohol. They are classified on the basis of the rel- ative amounts of nitrogen and phosphorus which they contain. Lecithin, kephalin and sphingomyelin are the best known mem- bers of the group.
The cerebrosides, obtained chiefly from the brain, contain nitrogen, but no phosphorus. On hydrolysis they yield, among other things the carbohj^drate galactose or one of its derivatives. The only two substances definitely known to be individuals in this group, and not mixtures, are phrenosin and kerasin. Little is known of the functions of the cerebrosides, but their presence in the brain is sufficient to make them interesting.
Lecithin. — Lecithin, classed as a phosphatid, is one of the interesting compounds of this type. It is found in all cells, in greatest amount in egg yolk which contains about 10%. Lecithin is soluble in absolute alcohol, and in ether, but may be precipitated from the latter solution by adding acetone. On hydrolysis lecithin yields higher fatty acids, glycerine, phos- phoric acid and an organic base choline. It is believed to have the following formula, where B indicates a fatty acid residue.
FATS, PHOSPHATIDS, ETC.
89
0
II CH2O — C ~ R
0
II CHO — C — R
I
0 0 — CH2 — CH2 N (CH3)30H
\ / P
/ \ 0 OH
The functions of lecithin in the body are not perfectly under- stood. Recent studies have made it seem probable that much of the ''fat" of the tissues, and the blood is in reality lecithin. It appears that the body easily synthesizes lecithin, so that it is not a necessary constituent of the diet. In fact the lecithin of the food probably is hydrolyzed by the lipase in the intestine into its constituent parts. Undoubtedly the lecithin in the cells aids them in holding water. Various other functions have been suggested, but in most cases the evidence is not conclusive.
Cholesterol. — Cholesterol is a substance which resembles the fats in some of its physical properties, but has little relation to them chemically. It is widely distributed in nature, and is found in large amounts in the brain and nerve tissue. It oc- curs also in small amounts in the blood, and in the bile, from which it is occasionally deposited in gall stones, whence it is most easily obtained. Cholesterol is insoluble in water, acids, or alkalies. It is readily soluble in hot alcohol, in ether, chloro- form, benzol, etc. Cholesterol crystallizes from hot alcohol or other solvents, forming large colorless plates. It gives many color reactions. If a chloroform solution of cholesterol is care- fully treated with concentrated sulphuric acid so as to form a layer of acid at the bottom of the test tube, the chloroform solu- tion becomes a brilliant red, and the acid layer dark red with a
90 PHYSIOLOGICAL CHEMISTRY
green fluorescence. This test is known as Salkowski's test. The Lieberman-Burchard test is one of the best of the choles- terol tests. To 2 c.c. of a chloroform solution of cholesterol, 10 drops of acetic anhydride and 2 drops of concentrated sul- phuric acid are added. A violet color appears which quickly turns to a blue green.
Cholesterol is an important substance physiologically. Ap- parently it protects the red blood corpuscles from certain harm- ful substances. It also seems to exert a restraining influence on some of the cell enzymes, and thus may act as a regulator of some of the cell activities. Cholesterol and certain of its allied substances are important in giving the cell its property of re- taining the water which it contains. The presence of choles- terol in the brain in such large quantities points to the suggestion that it may have some important role in the functioning or properties of this most important organ. In short, there are doubtless numerous other roles played by cholesterol in the body, of which we know very little as yet.
Cholesterol contains neither nitrogen nor phosphorus, and is classed as a sterol. It probably belongs to the group of terpenes.
CHAPTER V PROTEINS
Introductory. — The group of the proteins is of great im-. portance in nature, both in plants and in animals. Members of this group are found in every living cell, and are necessary for the life of the organism. An animal may get on very well for a long time on a diet containing no carbohydrate or fat, but if fed on a diet containing no protein, the animal will die. Plants build up their own proteins from nitrogen compounds of the soil or air, but animals are dependent upon plants or other animals for the materials out of which they construct their proteins. Plant structures contain large amounts of carbohy- drates along with protein and other materials, but the tissues of animals are made up largely of proteins. Thus muscle, nerve, ligament, skin, bone, blood, lymph, hair, nails, feathers, eggs, etc., all contain much protein. Often it is their chief solid constituent. In plants we find stores of protein laid away in seeds, such as the grains, and in many other places.
Elementary Composition. — The members of the group of pro- teins differ very widely among themselves in many properties, but they are all alike in certain respects, as for example in their elementary composition, for all proteins contain carbon, hydrogen, oxygen and nitrogen. Some contain also sulphur and some phosphorus. In addition to these elements we find sometimes others, such as iron, iodine, copper, manganese, etc. The proteins show individual variations in composition, but an average percentage is as follows:
C 50% N 16%
H 7% S 0.3%
0 22% P 0.4%
91
92
PHYSIOLOGICAL CHEMISTRY
The carbon forms the backbone of the protein as long chains to which the other elements are attached. Oxygen is present in a proportion smaller than in the carbohydrates, but greater than in tl>e fats. Nitrogen is present in various groupings, chiefly as imid — NH — groups which form the links holding the various parts of the protein together ; as amino groups — NHg ; and as — CONHg. These last two forms represent only a small part of the total nitrogen. Sulphur is present for the most part in unoxidized form — S — in one or two of the constitu- ents of the protein molecule, but may be present also in oxidized form. Phosphorus is present perhaps in different forms, chiefly in oxidized form as phosphate.
Classification. — A knowledge of the percentage composition such as that given above affords us no clue to the structure of the protein molecule, which is very complex. Our knowledge of the actual structure of the proteins is so limited that a classi- fication of the group has been worked out chiefly along other lines, making use of solubilities, source, etc., to distinguish groups, in connection with knowledge of chemical components so far as such information was available. The classification adopted by the American Society of Biological Chemists is as follows :
I. Simple Proteins.
1. Albumins
2. Globulins
3. Glutelins
4. Prolamines
5. Albuminoids
6. Histones
7. Protamines
II.
Conjugated
TEINS.
1. Glycoproteins
2. Phosphoproteins
3. Hemoglobins
4. Nucleoproteins
5. Lecithoproteins
Pro- III. Derived Proteins.
A. Primary Protein De- rivatives.
1. Proteans
2. Metaproteins
3. Coagulated P r o- teins
B. Secondary Protein Derivatives
1. Proteoses
2. Peptones
3. Peptids
This classification is by no means final or ideal. Compounds are known which do not fall well into any group, whereas others seem to be intermediate between two groups. This class- ification serves very well, however, to bring comparative order
PROTEINS 93
out of chaos until more is known of the structural differences which differentiate the various members of the group. Another classification, in use by English biochemists, differs only slight- ly from that given above. Hemoglobins are called chromopro- teins ; albuminoids, scleroproteins ; and the third division is sub- divided somewhat differently in the English classification.
Preparation of Proteins from Materials in Which They Oc- cur.— Most proteins occur naturally mixed with a large number of other materials. Only a few are found in a fairly pure state in nature. For purposes of study it is desirable to get them as free as possible from other compounds. Various meth- ods are useful, according to the properties of the protein to be isolated and those of the other substances present in the mixture or tissue. But even at best the purification of a protein is a difficult task and when finished it is often uncertain whether or not the substance is contaminated by some other protein of very similar composition and properties or by other compounds. Plant proteins often may be dissolved out with 10% sodium chloride or with alcohol and separated from one another by dialyzing the salt, or by fractional precipitation. In preparing proteins from animal tissues, the process is made somewhat more difficult by the fact that such tissues contain larger amounts of autolytic enzymes. These have the property of breaking down the cell constituents or altering them if the tis- sues are allowed to stand for some time before the proteins are extracted. Also, it is difficult to free the proteins from the fatty or phosphatid constituents of the cell without altering the nature of the protein itself. Various solvents may be used, such as salt solutions, dilute acids or alkalies. A method which is very satisfactory consists in freezing the tissue quickly, reduc- ing it to a fine powder and drying while still frozen. This ma- terial then may be extracted with petroleum ether to remove fats, phosphatids, cholesterol, etc. The residue is extracted with 10% sodium chloride solution, and the various proteins sepa- rated by fractional precipitation.
Some of the proteins may be purified by re-crystallization.
94 PHYSIOLOGICAL CHEMISTRY
Molecular Weight. — The molecular weight of the proteins is very high. Estimation of the molecular weight has been at- tended by great difficulties, because of the practical impos- sibility of obtaining pure proteins, and because of the unstable nature of the proteins themselves. The molecular weight of these compounds is so great that they cause only a very slight lowering of the freezing point, so that this method of determin- ing molecular weight has given results of questionable reliabil- ity. The boiling point method cannot be used, as proteins coag- ulate on heating. Various other methods have been devised, however, and as fairly uniform results are obtainable by differ- ent methods of analysis, we may draw fairly accurate conclu- sions as to the molecular weights of members of the group. One of the methods employed consists in estimating the sulphur con- tent of the protein. If the substance contains 0.5% sulphur then sulphur makes up 1/200 of the protein, and the molecular weight will be 200 times that of the sulphur present. If there are two sulphur atoms in the protein, they have a molecular weight of 64 (2X32). The molecular weight of the protein will thus be 200X64=12,800. Calculated on this basis most of the proteins will have a molecular weight of from 14,000-16,000. Calculating the molecular weight in other ways, such as from the amount of oxygen some of the proteins will take up, gives practically identical figures. More direct methods (measure- ments of osmotic pressure, etc.) confirm these figures, so that we are justified in assuming the molecular weight of most pro- teins to be roughly in the neighborhood of 15,000, although some recent evidence makes it seem that perhaps these figures, after all, are far too high. Comparing this figure with the mole- cular weights of some familiar compounds such as hydrochloric acid (36-f-), sodium hydrate (40) and sodium chloride (58+), we get an idea of the relative hugeness of the protein molecule.
Hydrolysis. — Any consideration of the molecular structure of compounds having such enormous molecules would seem to present almost insurmountable obstacles. As the result of the work chiefly of Kossel, Emil Fischer, Emil Abderhalden, and
PROTEINS 95
Osborne, however, much light has been thrown on the subject. It has been found that proteins if boiled with concentrated acids, with alkalies, or if acted on by certain enzymes will break down ultimately into fairly simple chemical substances. These are the oc amino acids. On hydrolysis, all proteins are broken down into a mixture of these compounds, of which about twenty have been obtained from proteins. All proteins, what- ever their nature, yield these same compounds. All the amino acids are not obtained from every protein, but in general a pro- tein yields most of them. Many proteins lack one, two, three, or perhaps more, and a few proteins are made up of relatively few different amino acids. The analytical methods in use do not give products to the amount of 100% of the original pro- tein, for there are losses at various stages in the process. Usu- ally only about % of the original substance is accounted for. From some proteins 85-90% has been recovered, and in the case of salmine 110%, this apparently impossible result being accounted for by the taking up of water when the amino acid complexes are split up. It is easy to understand the causes for the difference in behavior of different proteins. Although they all are made up of practically the same amino acids, these are present in different proteins in widely varying proportions, and also undoubtedly are arranged or put together differently. The following is a list of the amino acids which thus far have been obtained by the hydrolysis of proteins. It is quite pos- sible that as time goes on others will be added.
Amino Acids Obtained by Hydrolyzing Protein
A. Monoamino — monocarboxylic acids.
1. GlycocoU NH^CH.COOH
2. Alanine CH3CHNH2COOH
3. oc Amino Butyric CHXH^CHNH^COOH
CH3
\
4. Valine CH — CHNH^COOH
CH,
96
PHYSIOLOGICAL CHEMISTRY
5. Caprine
6. Leucine
7. Isoleucine
8. Serine
9. Cy stein
10. Cystin
CH3CH2CH2 CH2CHNH2COOH CH,
\
(
CH CH,CHNH,COOH
CH,
CH3 — CHg
CH,
\
<
CH CHNH^COOH
CHoOH
I CHNH2
I COOH
CH^SH
CHNH2
I COOH
CH.,S — S CH9
I I
CHNH2 CHNH,
COOH COOH
B. Monoamine dicarboxylie acids.
11. COOH 12. COOH
CH2
CHNH.
I COOH
Aspartic
CH^
CH2
I CHNH2
COOH Glutamic
PROTEINS
97
C. Diamino-monocarboxylic acids 13. Lysine |
NH,
14. Arginine D. Cyclic compounds.
15. Phenylalanine
16. Tyrosine
17. Tryptophane
18. Histidine
CH2CH2CH2CH2CHCOOH
/NH3 C = NH \NH CH2CH2CH2CHNH2COOH
H C HC/ \C CH^CHNH^COOH
HC\/C C H H
H
C HC/ \C CH2CHNH2COOH
HOC\/C C H H
H C
HC/ \C— C— CH2CHNH2COOH
HcV/C\/C C N H H H
H
C — N
\
CH
/
C — N
I H CHo
CHNH,
I COOH
98 PHYSIOLOGICAL CHEMISTRY
|
H^C — CH2 |
|
|
19. Proline |
H^C C— COOH \/ H N H |
|
(OH) H^C — CH 1 1 |
|
|
20. Oxyproline |
H^C C.COOH \/ H N |
H
The position of the hydroxyl is uncertain.
General Properties and Reactions of Amino Acids. — Soluhil- ity, Taste, Optical Activity. — The amino acids obtained by hydrolysis of protein are crystalline substances, which are readily soluble in water, Avith the exception of cystine, which dissolves with difficulty in both cold and hot water, and of tyrosine, which is quite insoluble in cold water, but dissolves more readily in hot water. Solutions of monoamino-monocar- boxylic acids are neutral in reaction. Solutions of dicarboxylic acids are acid, and of diamino acids are alkaline. The solu- tions of monoamino-monocarboxylic acids in reality have both acid and basic properties and should properly be classed as am- photeric. They all dissolve in dilute acids or alkalies, except cystin, which is not readily soluble in dilute ammonia. The amino acids vary in taste. Glycocoll, alanine and caprine are sweet, leucine is tasteless and isoleucine is bitter. With the exception of glycocoll, all the amino acids are optically active and exist in two forms, a dextro- and a levorotatory. Usually only one of the two is found as a protein constituent, and this is more often the levorotatory variety. If proteins are broken down by hydrolysis with alkali, the resulting amino acids are racemic, that is, they exist as equal amounts of the two optical isomers. The form of the acids not present in the protein is tjejieved to be prod aeed daring the hydrolysis. Hydrolysis by
PROTEINS 99
enzymes, and to a certain extent by acids produces optically active acids, either dextro- or levorotatory, but does not cause racemization.
Acids, Bases. — The amino acids add on acids such as hydro- chloric at the amino group. Thus
H
/
— NH2 + HCl-> — N = H2
CI
The acid raises the valence of the nitrogent to five. They also interact with bases to form salts.
— COOH + NaOH -> — COONa + H^O
At the amino group the amino acids add on the salts of certain metals such as cupric chloride, mercuric chloride, etc. This property is often made use of to precipitate amino acids.
Formaldehyde. — ^With formaldehyde, amino acids form meth- ylene compounds. The basic properties of the amino group are thus greatly reduced, and the terminal earboxyl group can be titrated with a standard alkali. On this process a much used method for estimating amino acids is based (Sorensen's method) .
I !
HC — NH2 + H2CO -> HC — N = CII2 + H2O
COOH COOH
Carhamino Reaction. — The amino acids interact with carbon dioxide in the presence of calcium salts to form carbamino com- pounds. These have the following structure:
R_CH — NH — C = 0
I I
0 = C — 0 — Ca — 0
If the nitrogen in this compound is determined, and also the amount of COj, which is combined, a relationship between the
100 PHYSIOLOGICAL CHEMISTRY
amounts of nitrogen and COg may be established. In case the
CO radicle R above contains no nitrogen -z^ = 1 for the above com- pound. If the radicle R contains nitrogen, however, as will be the case if the compound contains diamino acids, or is a sub- stance known as a peptid, in which two or more amino acids are
CO linked together, ^^ will be less than 1.
Oxidation. — Oxidation of amino acids may yield a variety of products according to the strength of the oxidizing agent. The NH, groups are not split off by acids or by alkalies to any ex- tent. Alkalies, however, split arginine into ornithine and urea, and split off the sulphur from cystin and cystein. The small amount of ammonia given off in acid hydrolysis of pro- tein is believed to come from the few acid amid groups — CO — NHg present. Oxidation with permanganate or hydro- gen peroxide yields pyruvic acid from alanine. This compound is interesting since it is believed to be one of the steps in the breaking down of carbohydrates in the body, and thus may be a substance by way of" which amino acids and monosaccharides can be converted into one another in the body. The substance has the formula
CH3
C = 0
I COOH
Nitrous Acid. — ^With nitrous acid the oc amino acids are broken down. Their nitrogen is liberated in the form of the free gas. This will be recognized as the familiar reaction of nitrous acid with primary amines.
R NH2 + HONO -^ ROH + N2 + H2O
This reaction is the basis for the Van Slyke method for estimat- ing amino acids, a method which has proved most useful in helping to settle some of the difficult questions with reference to the fate of the proteins in the body. From this brief re-
PROTEINS 101
view of some of the important facts relating to the amino acids we will turn our attention to the general properties and reac- tions of the proteins themselves.
General Protein Reactions
The general protein tests may be divided into two groups, the color tests and the precipitation tests.
Color Tests. — The protein color tests are by no means specific for proteins. They are tests which are given by certain group- ings or certain constituents generally found in the protein mole- cule. If the grouping upon which a test depends is absent from a particular protein, that protein will not respond to the test in question. Thus a single positive test should not be taken as evidence of the presence of a protein, but should be confirmed by some other test.
Biuret Test. — If concentrated sodium hydroxide is added to a protein solution, and then a few drops of very dilute copper sulphate solution, a violet color appears either at room tem- perature or on slight warming. This test is named from the fact that it is given by a substance biuret which is made from two molecules of urea with loss of NH,
|
NH2 |
|
|
\ |
|
|
NH2 |
C = 0 |
|
/ |
/ |
|
2 CO -^ |
HN |
|
\ |
\ |
|
NHo |
C = 0 |
|
/ |
|
|
NH2 |
|
|
Biuret |
This substance does not occur as a constituent of the protein molecule. The test depends on the presence of two amid groups — CO — -NHg united either directly or by a carbon or nitrogen atom. One of the amino groups may be substituted as — CO— NHR but not both of them. If the NHg of the amid
102 PHYSIOLOGICAL CHEMISTRY
groups is split off by the action of strong acids, the proteins will no longer give a biuret reaction. Ammonium salts such as am- monium sulphate interfere with the test, and should be decom- posed by boiling the mixture with strong alkali before making the test. Some of the proteins, e.g., the histones, and some of the products of protein hydrolysis give a reddish color.
Millon's Test. — If a few drops of Millon's reagent (a solution made by dissolving mercury in concentrated nitric acid) is added to a protein solution, a yellowish preciptate forms. On boiling, this precipitate turns rose pink. If the boiling is con- tinued the pink color is destroyed and the precipitate turns brown. At times the whole solution becomes pink. If the protein happens to be insoluble, it will give the test quite as well, turning a very decided pink or red. The test depends on the presence in the protein of tyrosine, and is given only by those proteins which contain this amino acid. Phenol or any other compound which contains a hydroxyphenyl group will give the test, however. The dihydroxybenzenes do not give the test unless one hydroxyl group is substituted. Chlorides, alco- hol or hydrogen peroxide will interfere with the test. Thus it is not serviceable to test urine for protein, since urine contains large amounts of sodium chloride.
Xanthoproteic Test. — If a protein is warmed with concen- trated nitric acid the mixture turns lemon yellow. On the ad- dition of alkali the color changes to a deep orange. The pro- tein need not be in solution. Students of chemistry will recall having performed this test upon themselves by getting con- centrated nitric acid upon the fingers, producing the familiar yellow spots. The skin is made of protein material. This test depends upon protein constituents containing the benzene ring, namely tyrosine, phenylalanine and tryptophane. The colored substance is a nitro derivative of benzene. Any substance, pro- tein or otherwise, containing a benzene ring will respond to this test.
Adamkiewicz or Hopkins-Cole Test. — If glacial acetic acid, or a solution of glyoxylic acid (prepared by reducing oxalic
PROTEINS 103
acid with magnesium or sodium amalgam) is added to a pro- tein solution, and concentrated sulphuric acid added so as to form a layer at the bottom of the test tube, a violet ring will form at the juncture of the two liquids. This test is due to the tryptophane in the protein molecule, and only those proteins containing this amino acid will respond to the test. When glacial acetic acid is used, the test is believed to be due to the presence as an impurity of glyoxylic acid, HCO — COOH, or other aldehydes. Nitrates, nitrites, chlorides, chlorates and some other substances interfere with this test.
Sulphur Test. — If concentrated sodium hydroxide and lead acetate are added to a protein solution and the mixture boiled, a brown or black color appears. The unoxidized sulphur of the cystein or cystin is split ofP and combines with the lead to form the dark brown or black lead sulphide.
Precipitation Reactions. — Proteins may be precipitated by a variety of reagents. The behavior of protein solutions with precipitation reagents, and in fact many other properties of protein solutions indicate that the proteins do not form true solutions such as that of sodium chloride in water. They form colloidal solutions.
Heat. — Many proteins are precipitated by heat. A slightly acid reaction, and the presence of salts is desirable if precipi- tation is to be complete. Alkaline solutions of proteins do not precipitate on boiling. Neutral solutions, especially if salts have been removed by dialysis, will precipitate only imperfect- ly. Solutions of some proteins, i.e., casein, gelatine and sec- ondary derived proteins such as proteoses and peptones do not precipitate on boiling. In general for each protein there is a specific precipitation temperature, but experimental conditions such as the amount of salts present, the rapidity of heating and other factors cause rather wide variations in the precipitation temperature. A protein precipitated by boiling in weak acid solution cannot readily be redissolved. Some as yet unknown change has taken place in the protein molecule, and the sub-
104 PHYSIOLOGICAL CHEMISTRY
stance is said to be coagulated. Proteins may be coagulated also in other ways.
Mineral Acids. — Proteins are precipitated by the addition of small amounts of the strong mineral acids, — hydrochloric, sulphuric and nitric. The precipitate dissolves in excess of the acid, particularly if the solution is heated. Glacial acetic acid does not precipitate proteins. There has been much discussion of the nature of this precipitation. Probably the proteins are thrown down in the form of salts. Precipitation with concen- trated nitric acid is often used as a test for proteins. To the solution to be tested, concentrated nitric acid is added carefully so that it will form a layer at the bottom of the test tube. In the presence of protein a cloudy ring appears at the juncture of the two liquids.
Salts. — Proteins are precipitated by salts. The salts of heavy metals, such as copper, iron, mercury, lead, etc., will throw down the proteins from their solutions. The precipi- tates formed are in many cases true salts of the protein and the metal. Where this is the case, the precipitation takes place best usually in a weakly alkaline solution, for in this condition the proteins are negatively charged, and will combine with the positive metal ions. Some proteins, such as protamines and his- tones, which have large amounts of diamino acids, form alkaline solutions and the protein carries positive charges. More alkali must be added to give these proteins negative charges than in the case of albumins. Casein, which contains much dicarboxylic acid (glutamic) carries negative charges (since it gives off hydrogen-ions to the water). Casein may thus be precipitated by metals even in slightly acid solution.
The conditions governing precipitation of proteins by metals are somewhat complicated by the fact that some metals such as mercury, gold, copper, and others (these metals have a lower solution tension than hydrogen) combine with the amino group also, so that salts of these metals will precipitate proteins in weakly acid as well as in weakly alkaline solution.
Three salts much used to precipitate proteins are ammonium
PROTEINS 105
sulphate, magnesium sulphate and sodium chloride. High and varying concentrations of these salts are necessary to throw down the different proteins. By the use of suitable amounts of these salts some of the protein groups may be separated from others. The process is known as "salting out." The proteins are not coagulated, and may be redissolved on removal of the salt.
Alkaloidal Reagents. — Many compounds known as alkaloidal reagents will precipitate proteins. Among these are several acids such as tannic, picric, phosphotungstic, phosphomolybdic, ferrocyanic, chromic, and dichromic. The precipitates undoubt- edly are compounds of protein with the negative-ion of the pre- cipitating reagent. The tests are thus carried out to best ad- vantage in weakly acid solution. Exceptions to this statement depend upon conditions similar to those discussed under pre- cipitation by the addition of salts.
Tungstic acid as a protein precipitant is of particular in- terest, since it is used to precipitate the proteins of the blood in the preparation of the protein-free blood filtrate used in the Folin and Wu system of blood analysis. Sodium tungstate is added to the diluted blood, and then sulphuric acid. Tungstic acid is set free, and precipitates the blood proteins completely, so that the filtrate is water-clear, and free from all protein.
Alcohol. — Alcohol in sufficient concentration will precipi- tate many of the proteins. Some few are soluble in alcohol, however. If the precipitate is allowed to stand in the alco- hol, it will become coagulated, and cannot be redissolved on re- moval of the alcohol.
Structure of the Protein Molecule
Since proteins make up so large a part of living tissue, and are indispensable to life it would be of great interest to find out, how the protein molecule is constructed. This problem has been studied by some of the foremost biochemists for a long time, and although the formula for a protein is still unknown, much is now known as to the manner in which the parts of a protein
106 PHYSIOLOGICAL CHEMISTRY
molecule are put together. On hydrolysis, proteins yield a mix- ture of amino acids. These, then, must be the building stones out of which the proteins are constructed. How are these building stones put together? Much evidence has accumulated to show that the amino acids are joined together in long chains, the different units being linked by what is known as the amid or ''peptid'* linking, — the union, with loss of water, of the car- boxyl group of one acid with the amino group of another.
NH2CH2COOH -f NH2CH2COOH -^ NH2CH2CO — NH — CH2COOH + H2O
A compound of this type is called a peptid, — if made from two amino acids, a dipeptid, if from three amino acids, a tripeptid, if from many amino acids, a polypeptid. On partial hydrolysis of proteins, such compounds actually have been found in the resulting mixture, and have been shown to be identical with compounds of known structure built up in the laboratory. The most complex compound of this nature yet synthesized was prepared by Emil Fischer. He prepared a substance having eighteen amino acids in the chain, thus an octadecapeptid. This compound had a molecular weight of 1213, and from its prop- erties, was still much simpler than a protein. The synthesis of such compounds is extremely laborious and expensive, so that no attempt has been made to carry the process further. One of the factors tending to increase the labor of synthesis is the fact that the amino acids used are optically active. As ordi- narily obtained, they consist of equal portions of the dextro- and levorotatory forms, and must be separated into the two varieties. The method most used* to accomplish this consists in preparing the salts of the two forms with some optically ac- tive base, such as brucine, cinchonine, etc. The brucine com- pounds of the d- and 1-forms may be separated since they usu- ally vary considerably in solubility, and on concentration one or the other will crystallize out first. A second method consists in allowing various microorganisms to act on the mixture of the two optical isomers. Usually one form will be destroyed
PROTEINS 107
by the microorganism, the other to a much smaller degree, or not at all.
The optically active acids then may be built up into peptids at will. Three methods are in use to effect this synthesis.
1. Ester metJtod. —
Glycocoll ester is converted into a ring compound
|
C2H5O 0 C CH2NH2 1 |
|
|
+ |
-> 2C2H5OH + |
|
H2N CH2COOC2H5 |
|
|
CH2 — NH |
|
|
/ |
\ |
|
CO |
CO |
|
\ |
/ |
|
NH — CH2 |
Under the action of alkali this ring compound is split open, forming a dipeptid
NH2CH2CO NH CH2COOH
Of course the amino acids used may be varied, and thus differ- ent dipeptids obtained. The method serves only to build up dipeptids, however, and if two different amino acids are used it has certain other disadvantages.
2. Synthesis by means of acid chlorides. — If glycocoll is treated with chloracetyl-chloride the following reaction takes place:
CICH2CO CI + NH2CH2COOH -> HCl + CI CH2CO — NH. CH2COOH
By the action of ammonia, the chlorine atom may be replaced by NHg, and a dipeptid results.
NH2CH2CO — NH. CH2COOH.
This may be treated with chloracetyl chloride again, and in a manner perfectly analagous to the first reaction, a tri-peptid or higher peptid built up. Here again various amino acids, and various halogen derivatives may be used. The limitation of this
108 PHYSIOLOGICAL CHEMISTRY
method lies in the fact that the chain can be extended only at one end.
3. By treating an amino acid or a peptid witJi pJiospJiorus pentacJiloride, the carboxyl group is converted into an acid chloride group which then may be caused to interact with the amino group of an amino acid or a peptid in a manner analagous to that described in the second method of synthesis. Thus gly- cocoll will be converted into NHgCHgCO CI which may be com- bined with a second glycocoU molecule or other amino acid to form a dipeptid as in 2.
The compounds prepared in these ways show many of the reactions characteristic of the products of partial hydrolysis of proteins, — thus some give the biuret reaction, those containing tyrosine give the Millon test, those containing tryptophane give the Adamkiewicz test, and certain of the more complex of them are precipitated by some of the protein precipitants. Many of them even are split by enzymes found in the body which are known to split protein decomposition products. These synthetic products thus are identical with those obtained in protein hy- drolysis, a fact which is one of the strongest pieces of evidence that the' proteins are constructed on the general plan of the polypeptids. Other evidence for this form of linkage in the protein is given by various facts. The proteins react only to a very limited extent with reagents which interact with free NHg groups. Very little hydrochloric acid is taken up by them, the formaldehyde reaction described above shows relatively few free amino groups, as does also the Van Slyke method also de- scribed above. In alkaline hydrolysis the amino acids become racemic for the most part. Only those whose carboxyl groups are combined, as is the case in the amid linking, will become racemic on hydrolysis. All these results are explained if the amino acids making up the proteins are assumed to be united by the "peptid linking. '^
The behavior of polypeptids with enzymes is especially in- teresting, and demonstrates the extremely specific character of enzyme action. For example, it has been shown that the di-
PROTEINS 109
peptid d-alanyl-d-alanine is split by pancreatic juice, but not d-alanyl-1-alanine. In general, those peptids made up of the optical form of the amino acids which is found in nature will be split by enzymes. Peptids made from the optical isomers which do not occur in nature are not split, or are split less readily by enzymes.
From a study of the polypeptids it has been learned that the ease with which a given polypeptid may be precipitated from solution depends not only on the size of the molecule, but also upon the particular amino acids present in it. Thus a poly- peptid containing tyrosine, tryptophane or cystin will pre- cipitate at a much lower concentration of a given precipitating reagent than a second polypeptid of equal molecular weight, containing none of these acids. Many polypeptids, both those prepared synthetically and those obtained from protein hydro- lysis will give characteristic protein reactions, though no poly- peptid has been prepared which closely approaches the pro- teins in the complexity and size of its molecule.
On hydrolizing a protein, that is, breaking it down into sim- pler products, the process evidently is not a symmetrical divi- sion and redivision of the molecule. In tryptic digestion tyro- sine, tryptophane and cystine are split off much quicker than alanine, leucine and valine, whereas glycocoll, proline and phenylalanine are not split off even after prolonged digestion. We must assume, therefore, that certain groups or ''building stones," are split off from the great protein molecule, which is thus reduced through various stages to proteoses, peptones and in complete hydrolysis to the amino acids.
Putrefaction of Proteins. — When proteins are broken down by the action of bacteria in the intestines, products are formed some of which are very injurious to the organism. The amino acids produced by the hydrolysis of the proteins are attacked and partially broken down in a variety of ways. From trypto- phane by the removal of its side chain, skatol and indol are produced.
110 PHYSIOLOGICAL CHEMISTRY
H H
C C
//\ //\
HC C — CCH3 HC C — CH
I II II I II II
HC C CH HC C CH
\/\/ \/\/ .
C N ON
H H H H
Skatol Indol
Prom amino acids, by removal of CO2 from the earboxyl group various amines are produced, e.g., from tyrosine, tyramine
H H
C C
// \ // \
HC C CH2 CH NH2 COOH HC C CH^ CH^ NH^
I II • I 11
HOC CH ->HOC CH
\ / \ /
c c
H H
From histidine, histamine is produced in a similar way. Such compounds are known by the name ''ptomaines," and they are extremely toxic. Cystine and cystein give rise to hydrogen sul- phide and other sulphur compounds. A condition of constipa- tion, in which intestinal contents are retained unduly long in the body, and putrefaction thus prolonged, favors the produc- tion of such substances. On absorption into the blood they give rise to headaches, general debility and in time, to more serious disorders. Undoubtedly certain compounds of this type are produced normally by the enzymes of the tissue cells them- selves, and it is probable that some of them are of importance for the regulation of certain body processes. Adrenaline is such a substance.
Individual Groups. Simple Proteins
The important characteristics of the proteins as a class have been considered. Let us now review the properties of groups and of individual proteins.
PROTEINS 111
Albumins. — The albumins are found widely distributed in nature. Serum albumin in the blood, ovalbumin in egg-white, lactalbumin in milk and myogen in muscle are the best known members of this group. Compounds resembling the albumins have been found in plants, although they differ in some of their properties from the animal albumins. Albumins contain no glycocoll, and relatively much sulphur (about 1.6-2.2%). They are soluble in water and dilute salt solutions, are precipitated by strong mineral acids, by saturation with ammonium sulphate in neutral solution and by many other precipitants. They are not precipitated by saturating a neutral solution with mag- nesium sulphate or sodium chloride, but if such a solution is acidified, the albumins precipitate. Albumins in solution coagu- late on boiling, if salts are present and if the solution is faintly acid.
Globulins. — Globulins also are widely distributed in nature, both in animals and in plants. They often are associated with albumins. Important members of the group are serum globu- lin, fibrinogen and its derivative fibrin of the blood, and myosin of the muscles, ovoglobulin in eggs, lactoglobulin in milk, neuro- globulin in nerve tissue, and several plant globulins such as edestin from hemp seed, legumin from peas, lentils, etc., and various others from nuts or other materials.
The globulins are insoluble in pure water, and they may be precipitated by pouring a solution of a globulin into a large volume of pure water, or by dialyzing out the salts against dis- tilled water through a parchment membrane. Globulins also differ from albumins in containing glycocoll, and in the greater ease with which they precipitate on the addition of a neutral salt. Thus they are precipitated by half saturation with am- monium sulphate or by saturation with magnesium sulphate or sodium chloride in neutral solution.
Fibrinogen has the property of clotting or coagulating, and is responsible for the clotting of the blood. If freshly drawn blood is stirred or beaten, the fibrinogen collects in stringy masses called fibrin. If washed free from corpuscles fibrin is a white,
112 PHYSIOLOGICAL CHEMISTRY
tough, elastic material which gives the usual protein tests. Myosinogen and myosin are the chief proteins of muscle tissue.
Glutelins. — The glutelins are proteins found only in the the seeds of plants. The important members of the group are glu- tenin from wheat, oryzenine from rice, and avenine from oats. There probably are others. They are characterized by being in- soluble in pure water, salt solutions or alcohol. They dissolve in dilute acid or alkali.
Prolamines. — The prolamines are proteins found only in the grains. They have been obtained from all grains except rice. The best known members are gliadin from wheat, zein from corn, and hordein from barley. The group was named because of their high content of proline. The prolamines are characterized by being soluble in 70-80% alcohol. They are insoluble in water. They contain only small amounts of arginine and histidine and no lysine.
Albuminoids. — Albuminoids are proteins obtained from a wide variety of sources in the animal world. Important mem- bers of the group are keratin from horn, nails, hair, hoofs, etc., collagen from connective tissue and bone, its derivative gelatine, which properly does not belong in this class however, and elastin, from ligaments and connective tissue. There also are various other albuminoids. The members of the group are classed to- gether for convenience, since they are insoluble in water and most protein solvents, and are constituents of protective or sup- porting portions of the body. Keratin is the chief constituent of hair, horn, nails, feathers, the epidermal layer of the skin, etc. A keratin also has been obtained from the brain and nerve tissue. There probably are several keratins. The keratins con- tain much sulphur (1-5%). They give the characteristic protein color tests.
Collagen is the chief constituent of the connective tissue and the chief organic constituent of bone. It occurs also in cartilage. There probably are several collagens. Collagen, if boiled with water or dilute acid, is converted into gelatine. Collagen is in- soluble in water, dilute salt solutions, dilute acids and alkalies.
PROTEINS 113
Gelatine, obtained by boiling collagen, swells up in cold water, but dissolves in hot. If sufficiently concentrated, such a solution will set, on cooling, to a jelly. Gelatine is not coagulated by boiling, nor will it precipitate on the addition of strong mineral acids or metallic salts. It may be precipitated by a variety of reagents, however, such as alcohol, tannic acid, etc. Gelatine gives the biuret reaction, but it contains neither tyrosine nor tryptophane, and thus if pure will not give the Millon or Hopkins-Cole tests. Gelatine contains much glycocoll. After prolonged boiling, a gelatine solution will no longer gel on cooling.
Elastin. — Elastin occurs in connective tissue, and in largest amount in the cervical ligament. Fresh elastin forms yellow- ish shreds or strings which are elastic in character. It is in- soluble in water and most of the protein solvents. Elastin con- tains large amounts of glycocoll and leucine. It does not give the Hopkins-Cole test.
Histones. — The histones are found chiefly in the sperma- tozoa of fish, but the globin portion of the hemoglobin of the blood is usually classed in this group. Their chief character- istic is a high percentage of diamino acids. In this respect they stand midway between the protamines and the remaining simple proteins. They are basic in nature. The group is not sharply defined.
Protajnines. — The protamines are the most basic of the pro- teins. They occur in the ripe spermatozoa of fish. They are named from their origin, as salmine, from the salmon, sturine from the sturgeon, etc. They are characterized by their high percentage of diamino acids, particularly arginine. Salmine contains 87% of arginine. As a result, solutions of protamines in water are alkaline in reaction. They give the biuret test, but most of them do not give the other color tests for proteins. They are precipitated fairly well by neutral, salts and are not coagulated by boiling.
Conjugated Proteins
The members of this group are made up of protein combined with some other non-protein substance, which is called the pros-
114 PHYSIOLOGICAL CHEMISTRY
thetic group. Among the conjugated proteins are substances of great biological importance.
Glycoproteins. — The glycoproteins are compounds which on breaking down yield a protein and a relatively high percentage of a carbohydrate or carbohydrate derivative. Other proteins also yield carbohydrates on hydrolysis, but in smaller amounts than the glycoproteins. The group is divided into two divisions, mucins and mucoids. They are very difficult to purify, espe- cially the mucins, since they are slimy in character, and as a result there is much disagreement as to their composition and properties. Mucin is secreted by the salivary glands, by certain mucous membranes and elsewhere. The skin of some of the lower animals secretes large quantities of mucin. The mucins are distinguished from the mucoids by their slimy character and by the fact that on precipitation with acetic acid, they do not re- dissolve in excess of acid. The mucoids are found in tendons, in cartilage, in the cornea and crystalline lens, in egg white and various other places. Some authors divide this group again into mucoids and chondroproteins. The mucoid of tendon or carti- lage may be extracted with lime water. On acidifying with acetic acid, the mucoid precipitates, but it will redissolve in ex- cess. Both the mucins and the mucoids contain a relatively high amount of sulphur, a part of which, at least, is in the form of oxidized sulphur. On hydrolysis the mucoids yield a substance chondroitic acid or chondroitin sulphuric acid, which has been the subject of much study. The carbohydrate radicle in the glycoproteins is usually glucosamine or galactosamine.
Phasphoproteins. — The phosphoproteinsi are compounds of importance because they furnish a large share of the protein nourishment of the growing young of animals and birds. There are two important members of the group, the casein of milk and the vitellin of egg yolk. They are characterized by containing a relatively high amount of phosphorus (about 1%) which is present in some form neither lecithin nor nucleic acid. On digestion, the phosphoproteins leave a difficultly digestible resi- due known as pseudonuclein. The phosphoproteins are acid in
PROTEINS 115
character. In their solubilities and precipitation properties they resemble both the globulins and the nucleoproteins. They may be distinguished from the former by their phosphorus content, and from the latter by the fact that they yield no purine bases on hydrolysis. Cow's milk contains about 3-4% casein, human milk about 0.5-1.5%. Casein is not coagulated by boiling. It is insoluble in water, but dissolves readily in dilute alkalies. From such a solution, and from milk, casein is precipitated by the addition of a small amount of acid. This occurs also in the souring of milk. Bacteria decompose the lactose, forming or- ganic acids. From this acid solution the casein is precipitated, causing the characteristic clotting of sour milk. It may be pre- pared by diluting milk, and adding a small amount of acetic acid. To free the casein from fat it may be redissolved in sodium carbonate and reprecipitated with acid. This process should be repeated several times if a pure product is desired. Casein contains no glycocoll, but little cystine and relatively much tryp- tophane. On hydrolysis it yields also several acids which are not obtained from other proteins. Casein is clotted by a fer- ment, rennin, which occurs in digesive juices, particularly the gastric juice. It may be well to state in this connection that the nomenclature of casein and its allied substances is somewhat confused. By many investigators the substance as it occurs in milk is called caseinogen. This is converted by rennin into another substance, paracasein. Paracasein differs from casein- ogen at least in one respect, the insolubility of its calcium salt. The calcium salt of caseinogen is soluble, that of paracasein in- soluble. Since calcium salts are found normally in milk, para- casein is precipitated as the familiar curdy material. If calcium is previously removed by adding an oxalate to the milk, the para- casein is formed from caseinogen by rennin, but will not precip- itate. Subsequent addition of excess of a soluble calcium salt will cause precipitation even if the milk has been boiled to de- stroy the rennin. In the transformation of caseinogen into para- casein a protein like substance called albumose, or ''whey pro- tein" is split off. This clotting is the first step in the digestion
116 PHYSIOLOGICAL CHEMISTRY
of casein in the stomach, and serves to prevent the milk from passing on too quickly into the intestine.
Vitellin. — Vitellin is found in egg yolk and may be prepared by extracting the yolk with ether to remove lecithin, cholesterol, etc. The residue is dissolved in salt solution and reprecipitated by pouring into a large volume of water. To remove the last of the lecithin it is necessary to extract with boiling alcohol.
Hemoglobins. — This group often is called the group of chro- moproteins, since its members usually are colored substances. The most important of them is hemoglobin or oxyhemoglobin, the red coloring matter of the blood. This compound is con- tained in the red corpuscles in higher animals. In some of the lower animals it is simply dissolved in the blood plasma. It, or a substance closely allied to it, occurs also in red muscle. The hemo- globin fulfils an important function in the body, that of trans- porting oxygen from the lungs to the tissues. If hemoglobin is exposed to a plentiful supply of oxygen, as is the case when the red corpuscles are passing through the capillaries surrounding the tiny air spaces, the alveola? in the lungs, it takes up oxygen and is converted into oxyhemoglobin. When the corpuscles con- taining this oxyhemoglobin reach the tissues, again they pass through a fine network of capillaries. Here, however, the oxygen supply is very low, as oxygen is rapidly used up by the cells in oxidation processes. The oxyhemoglobin gives up its oxygen, becoming hemoglobin again, and the oxygen passes out through the capillary walls to the region of low oxygen supply. Re- turning to the lungs, the hemoglobin takes up a fresh supply of oxygen, and again carries it to the tissues. This property of hemoglobin may be demonstrated easily as follows: On adding an equal volume of water to the blood, the hemoglobin diffuses out of the corpuscles and the solution becomes clear. The blood is said to be ''laked." If the mouth of the test tube is closed with the thumb or a stopper and the liquid shaken well, the color of the solution becomes a brilliant red. It now contains oxyhemoglobin, the color of which is a much brighter red than that of hemoglobin, this substance gives arterial blood its bright
PROTEINS 117
red color. If a mild reducing agent is added to this oxyhemoglo- bin the color becomes much darker. The oxygen has been taken away from the oxyhemoglobin, which has now become hemo- globin. This substance gives venous blood its dark red color. This process may be repeated indefinitely. A convenient mild reducing agent is Stokes' fluid which contains 2% ferrous sul- phate, 3% tartaric acid and ammonia sufficient to redissolve the precipitate which first forms on adding this reagent. The amount of oxygen which can be carried by a given amount of blood varies somewhat. On the average from 100 c.c. of fully oxygenated blood 19-20 c.c. of oxygen can be obtained (measured at 0° C. and 760 mm of mercury pressure) . A portion of this is simply dis- solved in the blood plasma, but most of it is combined with hemoglobin. The tissues do not remove all of the oxygen from the blood, for venous blood still contains considerable oxyhemo- globin. From 100 c.c. of venous blood an average of 15 c.c. of oxygen may be obtained, although this value varies considerably. The molecular weight of hemoglobin is very large, probably about 16,000, and one molecule of hemoglobin is believed to com- bine with one molecule of oxygen (Og). The air normally con- tains about 20% oxygen. This percentage may be much lowered, however, before the amount of oxyhemoglobin in a hemoglobin- oxyhcmoglobin mixture will be greatly diminished. If the per cent of oxygen is reduced to 13% of an atmosphere the mixture will still contain 93% oxyhemoglobin and only 7% hemoglobin.
The dissociation of oxyhemoglobin is favored by the presence of salts, carbon dioxide, and lactic acid. In the presence of any of these substances, or if their amounts are increased, oxyhemo- globin tends to give up some of its oxygen. The biological im- portance of this fact is evident. In the tissues, the production of carbon dioxide or lactic acid will aid in causing oxyhemo- globin to yield up its oxygen, which is then used in the various oxidation processes going on in the cell.
Hemoglobin may be split into two parts, globin, a protein, perhaps a histone, and hemochromogen. From oxyhemoglobin hematin is obtained in place of hemochromogen. Globin makes
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up about 9-5% of the hemoglobin, hemochromogen about 4%. The corpuscles contain about 30% of hemoglobin, an amount much greater than could be dissolved in an equal amount of fluid, so it must be assumed that hemoglobin is held in some loose combination with the other constituents of the corpuscles. Hemoglobin and many of its derivatives contain iron to the extent of about 3%. This iron seems to be directly connected with the ability of hemoglobin to combine with oxygen.
Oxyhemoglobin may be crystallized with comparative ease. Oxyhemoglobins from different animals form crystals of greatly varying form, and may be crystallized with varying degrees of ease. Those easiest to crystallize are oxyhemoglobin of the guinea pig, which forms tetrahedra, of the rat (rhomboids), of the squirrel (hexagons), of the horse, dog, etc. Oxyhemoglobin from man (long rods, rhomboids), ox, and other animals may be obtained in crystalline form, but with greater difficulty. Crys- tals of the types which crystallize easily may be obtained by a variety of methods. That of Rei chert consists in adding 1-5% of solid ammonium oxalate to blood, either before or after laking or defibrinating. A drop of this blood placed on a slide will crystallize quickly. On standing in the ice chest very beautiful large crystals form.
Reichert and Brown have studied the oxyhemoglobin crystals from the blood of a great many animals and maintain that bio- logical relationships may be traced in the crystal form, since oxyhemoglobins from animals of the same species crystallize in similar forms. Hemoglobin is more soluble than oxyhemoglobin, and is thus much more difficult to crystallize.
Detection of Hemoglobin. — In medical practice, and also in medico-legal cases it often is of the greatest importance to de- tect and identify hemoglobin or its derivatives. This is done in a variety of ways as follows.
Catalytic Activity of Blood. — If hydrogen peroxide is added to blood, or even to blood diluted to the point, where the solution is no longer colored, the peroxide is decomposed, and bubbles of oxygen gas are given off. If the blood is first boiled, no action
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takes place. .Thus it appears that the blood contains an enzyme capable of decomposing peroxides.
Hemin Test.— One of the best tests for blood is the prepara- tion of hemin crystals. Hemin is the hydrochloride of hema- tin. The hemin test will not differentiate between the blood of man and other animals. Although hemoglobins from different animals differ, it is the globin portion which varies, and the hematin from different animals is undoubtedly the same. The hemin test is very delicate. The suspected stains are extracted with water or weak alkali, the solution evaporated to dryness and the residue used for the test. If the solution is very dilute, the pigment may be precipitated with tannic acid, and the test made on the precipitate.
The test is performed by evaporating a drop of blood or sus- pected material to dryness on a slide, adding sodium chloride and a few drops of glacial acetic acid. The mixture is covered with a cover glass and heated carefully until the acid boils. On cool- ing, brown or reddish brown rhomboidal crystals of hemin form. If crystals do not form the first time, the mixture should be boiled again as above. It may be necessary to repeat the process several times. As hematin is very stable, blood stains which have putrefied, or which are even centuries old still will give the test. Wood smoke and swamp water do not destroy hem- atin ; it may be destroyed if certain moulds have grown on the stain however. It is of interest that the excreta spots of blood sucking insects will give a positive hemin test.
Guaiac Test. — Benzidine Test. — Blood has the property of transferring oxygen from hydrogen peroxide to easily oxidized chemical substances such as guaiac, benzidine and a variety of other compounds. On adding a small amount of a freshly pre- pared 1% alcoholic gum guaicum solution to a solution con- taining blood, and then a small volume* of 3% hydrogen peroxide, a bluish green color develops. Old turpentine which has not recently stood in direct sunlight may be used in place of the hydrogen peroxide. In performing the benzidine test it is best to dissolve in glacial acetic acid the amount of benzidine which
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can be taken on a knife point, then add an equal volume of hydrogen peroxide and the liquid to be tested. If blood is pres- ent, the color produced will be a greenish blue. If blood has been diluted 200,000 times it still will give a very definite posi- tive reaction. Other substances also respond to these tests, such as milk, living matter, etc. A slice of raw carrot gives a very good test. Milk, and living matter, (carrot) if boiled, no longer will give the test, whereas blood gives it quite as readily after boiling. Evidently in the case of blood the material responsible for the test is not an enzyme. As negative tests, these color tests may be taken to indicate the absence of blood. As positive tests, however, they are not conclusive without further con- firmation from other tests. Also they do not distinguish human blood from that of other animals.
Absorption Spectra of Oxyhemoglobin and Hemoglobin.— Hemoglobin and many of its derivatives show characteristic ab- sorption spectra. Thus a spectroscopic investigation often is of the greatest value in detecting blood in feces, urine, gastric contents or in stains in medico-legal work. The student is re- ferred to the discussion of absorption spectra under pentoses in the chapter on carbohydrates. The nature of the absorption bands depends not only upon the substance present, but the con- centration of the solution and thickness of the layer through which the light passes. Blood diluted ten times with water, and observed with a spectroscope in a flat sided cell about one centi- meter in thickness allows only a little red light to pass through. If the solution is diluted however, it shows two absorption bands near together between the D and E Frauenhofer lines. On fur- ther dilution these lines become fainter and at sufficient dilution, finally disappear.
If Stokes ' reagent is added to an oxyhemoglobin solution, thus converting the oxyhemoglobin into hemoglobin the double bands give place to a single continuous band in about the same loca- tion.
The amount of hemoglobin or oxyhemoglobin in blood or other fluids may be estimated by various means, the most con-
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venient consisting in comparing blood or diluted blood with a scale giving reds of different intensities corresponding to definite concentrations of hemoglobin.
Derivatives of the Hemoglobins. — Carbon Monoxide-Hemo- globin.— Hemoglobin forms compounds with various gases other than oxygen such as carbon monoxide, carbon dioxide, etc. Car- bon monoxide, which is a constituent of illuminating gas com- bines with hemoglobin in molecular proportions. Its union ap- parently is firmer than that of oxygen, and apparently it com- bines with hemoglobin at the same place as does oxygen, for if both gases are present, carbon monoxide hemoglobin is formed, and the taking up of oxygen is interfered with. The carbon monoxide may be removed, however, by passing a stream of air through the liquid for some time, and oxyhemoglobin will be formed. In cases of asphyxiation by illuminating gas, carbon monoxide is found in the blood, thus preventing the proper transportation of oxygen to the tissues.
Solutions of carbon monoxide hemoglobin are a bright cherry red. The absorption bands look much like those of oxyhemo- globin,— two dark bands between the D and the E lines. They are slightly nearer the violet end of the spectrum however, and on adding Stokes' reagent to the mixture they do not, as does the hemoglobin spectrum, give place to a single broad band.
Carbon dioxide apparently combines with hemoglobin at a point different from that at which oxygen combines, as combin- ing with one does not prevent hemoglobin from combining with the other also.
MetJiemoglobin. — Methemoglobin is a compound derived from oxyhemoglobin. It contains the same amount of oxygen as o*xy- hemoglobin, but the oxygen is more firmly united than in oxy- hemoglobin. Methemoglobin is found in the blood after poison- ing with chlorates, amyl nitrite, etc., and is found occasionally in the urine, in transudates, cystic fluids, and elsewhere. Out- side the body it may be prepared for study by adding fresh potassium ferricyanide solution, permanganate or other sub- stances to oxyhemoglobin solutions. The solution turns a muddy
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brown. On dilution and observation with the spectroscope, a dark absorption band between the C and D lines is observed. Two fainter bands in the position of the oxyhemoglobin bands are considered by some investigators to be due to the presence of a small amount of this latter pigment. On adding Stokes' reagent to a methemoglobin solution, the substance is changed first into oxyhemoglobin, and this into hemoglobin, with corresponding changes in the spectrum.
On adding an alkali to a methemoglobin solutio^, alkaline methemoglobin is formed, which gives a characteristic spectrum of its own.
Acid Hematin. — Hematin is the compound into which oxy- hemoglobin may be split by the action of acids