STUDIES IN THE PHYSICAL CHEMISTRY OF THE PROTEINS.

Transcription

1 STUDIES IN THE PHYSICAL CHEMISTRY OF THE PROTEINS. V. THE MOLECULAR WEIGHTS OF THE PROTEINS.* PART 1. THE MINIMAL MOLECULAR WEIGHTS OF CERTAIN PROTEINS. BY EDWIN J. COHN, JESSIE L. HENDRY, AND ADELA M. PRENTISS. (Prom the Department of Physical Chemistry in the Laboratories of Physiology, Harvard Medical School, Boston.) (Received for publication, February 11, 1925.) CONTENTS. PAoE I. Introduction Determination of molecular weights by means of osmotic pressuremeasurements Determination of minimal molecular weights by means of combining and containing weights II. Calculation of the minimal molecular weights of proteins from their composition Iron content of hemoglobin Copper content of hemocyanin Sulfur content of proteins Sulfide sulfur content of proteins Phosphorus content of proteins Tryptophane content of proteins Tyrosine and cystine contents of proteins Histidine, arginine, and lysine contents of proteins III. Calculation of the minimal molecular weights of proteins from their equivalent weights Saturation of proteins with gases % Saturation of proteins with acids or bases determined by electromotive force measurements Combination of proteins with acids and bases determined by solubility measurements * The results of this investigation were reported at the meeting of the American Society of Biological Chemists, at Washington, D. C., December, 1924 (11). 721

2 722 Physical Chemistry of Proteins. V IV. Minimal molecular weights of certain proteins IIemoglobin Hemocyanin Egg albumin Casein Zein Gliadin Glutenin Gelatin F,destin Serum globulin Serum albumin E ibrin Bence-Jones protein V. Discussion VI. Summary I. INTRODUCTION. The large molecular weights of the proteins must be considered one of their outst,anding characteristics, in great part responsible for such colloidal asp&s of their behaviour as their failure to pass through many natural and artificial membranes. It is probably because of their size that proteins are retained by cell walls through which their constituent parts and their decomposition products freely pass. The cellular structure of biological systems, and their stability under changing metabolic condit,ions, depends, therefore, upon the separat,ion and the concentration of large organic molecules within limiting membranes. Many natural plant and animal cells were utilized in the development of our modern theory of solutions, because of t.he permeability of their cell walls to small organic and inorganic molecules, and their impermeability to such of their constituents as the proteins. In the classical paper before the Swedish Academy in which van t Hoff applied the gas laws to solutions by demonstrating the relation between osmotic pressure and the number of dissolved molecules, the argument rested in part upon De Vries measurements of the plasmolysis of plant cells, in part upon Hamburger s measurements of the hemolysis of blood corpuscles, and in part upon Pfeffer s experiments with artificial

3 Cohn, Hendry, and Prentiss 723 membranes (72). In the former, natural cells served as osmometers; in the latter, artificial cells, impermeable to certain molecules, allowed of the direct measurement of the osmotic pressures they produced. Determination of Molecular Weights by Means of Osmotic Pressure Measurements.-Since natural and artificial cells could be used as delicate indicators of the osmotic pressure of solutions, it was natural to suppose that measurements of the osmotic pressures of such constituents of cells as the proteins might lead to exact knowledge of their molecular weights and volumes. Sorensen and his collaborators have recently determined the molecular weight of egg albumin (70), serum albumin, and serum globulin with great accuracy by measurements of osmotic pressure. These investigators took into account sources of error involved in such measurements, which were not completely understood by earlier workers. These depend upon the reactivity and the multivalent character of the proteins, and upon the dissociation of the different compounds that proteins form with each other and with acids, bases, and salts. As a result of these complications, measurements of the osmotic pressures of proteins have not always led to consistent estimates of their molecular weights. The measurements on hemoglobin solutions of Reid, Htifner and Gansser, Roaf, and of Adair illustrate this contention. Reid (66) prepared the haemoglobin by crystallisation and obtained a pressure of about 4 millitnetres of mercury for each 1 per cent. of haemoglobin 3 or a molecular weight of over 42,000. Hiifner and Gansser (41), on the other hand, found the molecular weight of horse hemoglobin to be approximately 15,000, and of ox hemoglobin 16,000. The great effect of the solvent upon the osmotic pressures of hemoglobin solutions was first indicated by Roaf (67), and has since been systematically studied for other proteins by Lillie (48), and for hemoglobin by Adair (3). In Roaf s investigation three deter- 1 Sorensen, S. P. L., personal communication. * Different compounds of the same protein exert different pressures both because of the different number of particles into which they dissociate, and because of the complicated membrane equilibria which arise whenever the memhrane is permeable to certain of the ions of such compounds, and impermeable to others (50). 3 Roaf (67), p. i.

4 724 Physical Chemistry of Proteins. V minations of osmotic pressure were made, the first with distilled water, the second with 0.34 per cent. sodium bicarbonate (0.04 N) and the third with 0.2 per cent. sodium carbonate (0.04N). The osmotic pressures obtained for 1 per cent. haemoglobin were 5.7, 5.3 and 11.6 millimctres of mercury respectively. These pressures correspond to aggregates of 29787, 32035, and It should be noted that Roaf s lowest value for the molecular weight of hemoglobin in the presence of sodium carbonate was of the same order as IIiifner and Gansser s value. In this case the haemoglobin aggregate corresponds to its molecular weight calculated from the amount of iron present.3 Where the molecular weight is higher, it has been assumed that the hemoglobin existed in aggregates. Lillie s (48), Sorensen s (70), and Loeb s (50) investigations of the osmotic pressures of other proteins render it more probable that the lowest pressures correspond to those produced by the uncombined protein, while the higher pressures represent the dissociation of protein compounds with their attendant membrane equilibria. Determination of Minimal Molecular Weights by imeans of Combining and Containing Weights.-With the development of the modern theory of solutions, it was inevitable that the measurement of the osmotic pressures of protein solutions should seem the best method for the determination of their molecular weights. In the last 30 years such investigations have revealed complications which depended upon phenomena that are only now beginning to be understood, and that have thus far been adequately considered in but few researches. Meanwhile there has accumulated a vast body of analytical information regarding the composition of proteins on the basis of which their minimal molecular weights can be calculated, and a large number of physicochemical methods on the basis of which their equivalent combining weights can bc determined. In the succeeding sections of this paper the evidence that can be derived from the simultaneous consideration of the analytical and physical chemistry of the proteins will be considered. In a subsequent communication the relative size of the molecules of a series of proteins will be determined by the method of dialysis or ultrafiltration (7), and their probable molecular weights estimated as integral multiples of their

5 Cohn, Hendry, and Prentiss 725 minimal molecular weights. This procedure of estimating molecular weights as multiples of minimal molecular weights has often been followed in classical inorganic and organic chemistry. II. Calculation of the Minimal MolecuLr Weights of Proteins from Their Composition. Whereas great uncertainty exists concerning the true molecular weight of hemoglobin, the equivalent combining weight of this protein, and consequently its minimal molecular weight has bsen accurately known for 30 years. In 1894 Hiifner (39) studied the carbon monoxide-combining capacity of the hemoglobin of the ox, and found that 16,721 gm. of hemoglobin combined with each mol of carbon monoxide. Oxygen combines with the same amount of hemoglobin as does carbon monoxide (63). It is therefore certain that these important physiological reactions proceed stoichiometrically. Iron Content of Henzoglobin.-Hemoglobin contains iron. Zinoffsky, Hiifner, Jaquet, and Abderhalden have determined the amount of iron contained in the hemoglobin of different species. Their results indicate that hemoglobin always contains between and 0.40 per cent of iron, and at the same time suggest the first indirect method of estimating the minimal molecular weight of a protein in terms of a well known constituent of its molecule. For if the per cent of iron in the hemoglobin molecule of the horse represents but 1 gm. atom of iron, the molecular weight of the protein must be approximately 16,669. The relation upon which calculations of this type depend is expressed by the equat,ion Minimal molecular weight of protein = ~~r~~n~~~~:~~~r~e~ (1) Precise est,imates of minimal molecular weights, based upon actual determinations of the iron content of the hemoglobins of different species, are contained in Table I. Copper Content of Hemocyanin.-The proteins of the blood of certain of the Mollusca and Arthropoda contain copper, which presumably subserves the same function as the iron of hemo-

6 726 Physical Chemistry of Proteins. V globin. These proteins, because of their blue color, are all termed hemocyanins. The different amounts of copper in the hemocyanins of different genera and phyla indicate molecular weights that arc not identical (64). Further investigation has demonstrated that these copper-containing proteins differ not only in their minimal molecular weights, but in many other respects (5). Sulfur Content of Proteins.-Hemoglobin, hemocyanin, and many other proteins contain sulfur. Analyses of the sulfur contents of proteins, therefore, offer another method of estimating their minimal molecular weights. In the case of hemoglobin and hemocyanin, consideration of the sulfur content has for the most part confirmed t.he molecular weights postulated on the basis of the iron analysis. Occasionally, however, as in the case of the sulfur content of the hemoglobin of the ox, the sulfur analysis suggests that the molecular weight may be at least twice that demanded by the iron analysis. Sulfide A%&u- Content of Proteins.-The large amount of sulfur that many proteins contain diminishes the usefulness of the analysis of total sulfur, for the smallest weights of proteins that can contain 1 atom of sulfur arc often too small a fraction of their minimal molecular weights to aid in their estimation. Part of the sulfur in the protein molecule is,. however, sulfide sulfur; estimated by means of its lead-blackening property (53). T. B. Osborne (58) has studied the ratio of sulfide sulfur to total sulfur in a series of proteins, and has shown that the sulfide sulfur usually represents an integral fraction of the total sulfur. On the basis of this relation, Osborne estimated that the molecular weights of many proteins were in the neighborhood of 30,000 (57). The sulfide sulfur in the protein molecule has been supposed to represent, at least in part, the sulfur either in the ammo acid cystine or cystcine. Cysteine contains but 1 atom of sulfur, while cystine contains 2. Whatever the nature of the sulfide sulfur, it represents an integral part of the protein molecule, and the quantities that analyses reveal can be used in estimating the minimal molecular weights of proteins, in the same manner as can their iron, copper, or total sulfur content. Phosphorus Content of Proteins.-Certain proteins contain small amounts of phosphorus. Casein contains both sulfur and phosphorus, and the amounts of these two elements have led to

7 Cohn, Hendry, and Prentiss 727 almost identical estimates of the minimal molecular weight of this protein at 4,220 and 4,372 respectively (47, 8). Tryptophane Content of Proteins.-The tryptophane content of casein suggests that its molecular weight must be some multiple of the minimal molecular weight deduced from its phosphorus or sulfur content. Tryptophane exists in the casein molecule to the extent of 1.5 per cent according to the earlier analysts (59), 1.54 per cent according to Folin and Looney (21), and 1.7 per cent according to Dakin (16). The lower figure substituted in a relation similar to equation (1) leads to a molecular weight of 13,606; the higher, to 12,006. The average, 12,806, is three times the weight calculated from the phosphorus or from the sulfur content of casein. A minimal molecular weight of this order enabled us, in a previous communication (14), to approximate the molecular composition of casein. This molecule contains but 2 molecules of histidine, 3 of arginine and phenylalanine, and 4 of aspartic acid and tyrosine. The amino acids present in larger amounts can also be calculated, but cannot be considered as giving further evidence regarding the size of the molecule. That the assumption of this number of amino acid molecules leads to a minimal molecular weight near 12,800 gm. is demonstrated by the calculation in Table IV. Tryptophane is present in many protein molecules to a smaller extent than nearly any amino acid except cystine. Its quantitative isolation and estimation have, however, been difficult. In recent years numerous calorimetric methods have been evolved for determining the amount of this amino acid, as well as of tyrosine and of cystine, in protein hydrolysates. These methods avoid the losses that occur in the separation and quantitative estimation of amino acids. The excellent agreement between the tryptophane content of casein, as estimated by Folin and Looney, and the yields that have been obtained by isolation has already been alluded to. Tyrosine and Cystine Contents of Proteins.-Folin and Looney (21) have estimated the tryptophane, tyrosine, and cystine contents of a large number of proteins, and a number of other investigators have estimated these amino acids in different proteins, either by these or by other calorimetric methods. Whether

8 728 Physical Chemistry of Proteins. V cysteine or cystine exists in the protein molecule is still a debated question, but the form that is usually isolated from protein hydrolysates is cystine. If cysteine is present in the protein, the minimal molecular weight would, of course, be half that calculated from the cystine analyses. Histidine, Arginine, and Lysine Contents of Proteins.--Van Slyke s (71) nitrogen distribution method of determining the diamino acids in proteins also avoids the losses often attendant upon the separation and gravimetric estimation of amino acids. Moreover, many proteins contain but a small number of molecules of either histidine, arginine, or lysine. Accordingly, we have recalculated his results from nitrogen as per cent of nitrogen to per cent of protein, and included them in our tabulation of significant data for the determination of molecular weights.4 The yields of certain amino acids, determined by the classical methods, have occasionally been included. The internal evidence offered by the data here presented is such that this procedure is less arbitrary than it may appear. For when the minimal molecular weights, calculated on the basis of a number of constituents of the same molecule, lead to values that are related to each other in definite and integral proportions, the results attain a significance which is greater than can be claimed for an individual analysis. Finally, if the equivalent combining weights of the proteins, determined by one of the physicochemical methods that we shall now consider, yield results that are also consistent with the minimal molecular weights, calculated by analytical methods, a body of evidence accumulates which can scarcely be considered fortuitous. III. Calculation of the M inimal Molecular Weights of Proteins from Their Equivalent Weights. Saturation of Proteins,with Gases.--ilny stoichiometric reaction involving a protein may be utilized in estimating its equivalent weight. From the equivalent combining weight of the 4 D. Jordan Lloyd (49) has previously attempted to determine the molecular weight of gelatin from Van Slyke s data. Her method was different from that here employed.

9 Cohn, Hendry, and Prentiss 729 reagent, the amount required to saturate or combine a given weight of the protein can be calculated by means of the relation Equivalent combining n-eight of protein Weight of combined protein Equivalent weight of reagent = iiightofcombined reagent G9 Certain proteins which are involved in the transport of oxygen in the body, such as the hemoglobins and the hemocyanins, combine chemically with oxygen, and some also with carbon monoxide. From the carbon monoxide-combining capacity of hemoglobin, Ilufner early estimated that its minimal molecular weight was 16,721 (39). His result was, as we have seen, in excellent agreement with the weight of hemoglobin that contained 1 atom of iron. As early as 1894 the minimal molecular mcight of a protein had, therefore, been determined by the simultaneous consideration of analytical and physicochemical evidence. Saturation of Proteins with Acids or Bases Determined by Electromotiz;e Force Measurements.-Whereas only certain specialized proteins combine with gases, all proteins combine with acids or with bases. The amount of acid or base with which a protein can combine depends in the last analysis upon its composition and structure (12, 13, 25). Without reference to the nature of the groups involved in these reactions, the amount of combination at saturation of the protein can be utilized for the calculation of its equivalent combining weight, by means of the relation expressed in equation (2). The electromotive force methods for determining the maximum acid- or base-combining capacity of proteins have been considered in detail by Robertson (68), Pauli (62), and more recently by Hitchcock (34, 35), by Greenberg and Schmidt (27), and by us (12). Robertson determined in this way the equivalent combining weight for base of cnsein, serum globulin, and ovomucoid. A recalculation of Hitchcock s potential measurements has yielded the combining capacities of gelatin and edestin. Greenberg and Schmidt have also studied the base-combining capacity of gelatin, casein, and gliadin. These results have been considered in calculating minimal molecular weights wherever the protein did not contain so many groups as to lead to too low an equivalent weight. Bracewell (9) also measured the acid-combining capacity of a few proteins in connection with his attempt to show that this

10 Physical Chemistry of Proteins. V property depended upon their lysine and arginine, but not upon their histidine content. His method was less accurate than the electrometric procedure that has generally been employed. The reason for this will be considered in another place in connection with a study of the relation between the diamino acids in proteins and their acid-combining capacity.6 For completeness we have often included Bracewell s values, and considered them in the estimation of the equivalent combining weights of the proteins. In the case of a few proteins the total number of acid or of basic groups is so low that their equivalent combining weights at saturation are of great value in the calculation of their minimal molecular weights. This is t,rue of the acid groups in zein, gliadin, gelatin, and egg albumin, and of the basic groups in gliadin. In most proteins, however, the number of acid or basic groups is so large as to render measurements of their acid- or base-combining capacities at saturation almost valueless for this purpose. Combination of Proteins with Acids and Bases Determined by Solubility Measurements.-Many proteins are very insoluble when uncombined with acid or base. Thus casein only dissolves in water to the extent of 0.11 gm., serum globulin to the extent of 0.07 gm. (II), and zein to the extent of 0.05 gm. in 1 liter (13). Certain of the globulins, the glutelins, and the prolamins, all of which are relatively insoluble in water, have very wide precipitation zones. These appear to form insoluble compounds with acids and with bases. This must occur in the case of proteins like zein and gliadin which do not pass into solution until very alkaline reactions are reached, but which nevertheless combine base at neutral reactions. It would appear that these proteins dissolve only after neutralization of all their base-combining groups. In one series of experiments with zein, the addition of two-thirds of the maximal base-combining capacity resulted in the solution of less than one-tenth of the protein present. The base must have combined with the zein to form one or more insoluble compounds. T. B. Osborne (56) as early as 1902 studied the amount of acid required to form an insoluble hydrochloride of neutral edestin. He showed that the combining weight of protein, as 6 Cohn, E. J., and Berggren, R. E. L., unpublished data.

11 Cohn, Hendry, and Prentiss 731 calculated from an analysis of the amount of acid in this compound, was a multiple of the minimal molecular weight calculated from its sulfur content. Here, again, the simultaneous consideration of the composition of a protein, and of its reactions, has yielded an accurate estimate of its minimal molecular weight. When slight,ly larger amounts of acid or of base are added to certain proteins in the neighborhood of their isoelectric points, an amount of protein passes into solution which is proportional to the base added. Measurements of the solubility of edestin in systems containing protein in excess and known concentrations of base or of acid were also made by Osborne (56).. More recently WC have studied the solubility of cascin and of serum globulin in such systems. The weight of protein dissolved in combination with the reagent can be calculated from the increased solubility produced by increments of base by means of equation (2). Only a few of the many free groups that such proteins contain usually dissociate in the neighborhood of their isoelectric points. Small amounts of reagent, therefore, combine with large amounts of the protein. As a result equivalent combining weights, calculated from solubility measurements, are of inestimable value in calculating the minimal molecular weights of slightly soluble proteins. Up to the present, the equivalent weights only of casein and of serum globulin have been accurately determined by the solubility method. Bence-Jones protein has also been studied to some extent, and the equivalent weights of other proteins should presently be available. Meanwhile it seemed desirable to report the methods that are being employed and the results that have thus far been obtained, together with the analytical information that has been collected. IV. Minimal Molecular Weights of Certnin Proteins. Hemoglobin.-Hemoglobin was the first protein whose minimal molecular weight was adequately determined. Moreover, five different methods have been employed in its estimation by a score of different investigators. In the latter part of the last century, Zinoffsky and, later, Hiifner, and Jaquet (42) studied the iron and sulfur contents of

12 732 Physical Chemistry of Proteins. V the hemoglobins of a large number of species. Their analyt,ical results are summarized in Table I. In all of the six species considered, the hemoglobin contained between and 0.40 per cent, of iron. Taking the atomic weight of iron as 55.54, the weight of protein containing 1 atom of iron has been calculated by means of the relation Atomic weight of iron X 100 Minimal molecular weight of Hb = -Percentf ;ron in Hb (3) These analytical results indicate that the minimal molecular weights of the hemoglobins are not identical. The hemoglobin of the pig appears to have the smallest minimal molecular weight, 13,960, and of the horse and fowl the largest, 16,669. The minimal molecular weights of the hemoglobins of the ox and of the dog are essentially identical to the latter, or 16,619. This difference is, of course, smaller than the experimental error, and it might therefore be argued that all hemoglobins had the same molecular weight, and that the higher percentage of iron in the cat and pig were due to experimental error. The sulfur contents of the hemoglobins of these same species not only give additional information regarding the minimal molecular weights of these proteins, but also demonstrate differences in the hemoglobins of different species quite as conclusively as the more recent investigations of Reichert and Brown (65) and of Landsteiner and Heidelbcrger (46). The weight of hemoglobin containing 1 atom of sulfur is in every case lower than the weight containing 1 atom of iron. As a result, the hemoglobin molecule must contain a larger number of atoms of sulfur than of iron. The ratio of iron atoms to sulfur atoms in the hemoglobin of the horse and of the pig is as 1:2. On the basis of 2 atoms of sulfur in horse hemoglobin, its molecular weight must be 16,878, which is in excellent agreement with the value calculated from its iron content, 16,669. The agreement in the case of pig hemoglobin is almost as satisfactory, although the minimal molecular weight would appear to be different. The ratio of iron atoms to sulfur atoms in the hemoglobin of the dog and of the cat is, however, as 1:3. The differences in these iron-sulfur ratios constitute conclusive evidence that the hemoglobins of these species cannot be identical.

13 Cohn, Hendry, and Prentiss 733 The analyses of Hiifner and of Jaquet, which have elsewhere led to such consistent results, do not yield a simple ratio of iron atoms to sulfur atoms in the hemoglobin of the ox or of the fowl, TABLE I. Minimal Molecular Weights of the Hemoglobins. Method. Horse. Pig. cat. ox. Fowl. Dog. Iron content (73). Sulfide sulfur (69). Sulfur I (73). Iron I Sulfur Iron Sulfur < CO-combining capacity (39). Iron content (39). Sulfur (33). (54). Arginine (71). Iron Sulfur I per ceni gm , , , , , ,446 (38) , ,960 (38) , ,362 ( 1) , ,954 ( 1) , , (42) ,669 (42) ,729 Iron (42) ,619 Sulfide sulfur (57) ,573 Sulfur (42) ,646 16, ,442 16, ,238 7, ,635 6, ,405 4, ,356 33,338 33,561 49,857 47,865 50,814 on the basis of a molecular weight in the neighborhood of 16,700.6 However, if it be assumed that the molecular weight is twice as great, or approximately 33,400, then the ratio of iron atoms to sulfur atoms in ox hemoglobin becomes 2:s and in fowl hemo- BMtiller (54) gives 0.48 per cent for the sulfur content of ox hemoglobin, referring presumably to an article of Htifner s (39). In an earlier article (38) Hiifner gives the sulfur content of ox hemoglobin as

14 734 Physical Chemistry of Proteins. V globin 2: 9. As a result, these.t,wo instances not only add further evidence regarding t,he specificity of the hemoglobins of different species, but indicate that the molecular weight of this protein must be greater than 16,700, at least in certain forms. The carbon monoxide-combining capacity of ox hemoglobin also led Hiifner to a minimal molecular weight of 16,721 for this protein. This value is almost identical with that derived from its iron content, and Peters (63) has since demonstrated that the oxygen-combining capacity of hemoglobin was also proportional to its iron content. The osmotic pressure determinations of Hiifner and Gansser (41) and of Roaf (67) that were carried out in alkaline solution indicated molecular weights of the order of 16,000. Roaf s determinations of the osmotic pressure of hemoglobin in distilled water and in sodium bicarbonate, however, indicated molecular weights of 29,787 and 32,035. Adair s recent measurements with horse and human hemoglobin, which have been corrected for the Donnan equilibrium, indicate still higher molecular weights. These results suggest, for reasons already indicated in the first sections of this paper, that the true molecular weight of hemoglobin is at least 33,400, and that the higher osmotic pressures that have occasionally led investigators to postulate lower molecular weights are to be explained as due either to dissociation of hemoglobin in alkaline solution, or to the membrane equilibria attending such dissociation. There is evidence, however, that the molecular weight, of certain hemoglobins may be higher than 33,400. Weymouth Reid (66) estimated the molecular weight of recrystallized dog hemoglobin by the osmotic pressure method. He obtained slightly higher osmotic pressures with twice recrystallized hemoglobin than with hemoglobin that had been recrystallized but once. Measurements upon the latter indicated a molecular weight of approximately 48,000, while his results with the twice recrystallized protein led to only a slightly lower value in the neighborhood of 42,000. Osborne (57) has determined the sulfide sulfur in the hemoglobin of the dog. His estimate made in 1902 one can probably rely upon.... as reasonably accurate. The smallest weight 7 Personal communication.

15 Cohn, Hendry, and Prentiss of dog hemoglobin that can, on the basis of this determination, contain 1 gm. atom of sulfide sulfur would be 9,573 gm. The smallest molecular weight that can be postulated for dog hemoglobin on the basis of the simultaneous consideration of both the iron and the sulfide sulfur contents is five times the latter value, or approximately 47,865, a result which is in good agreement with that calculated from Reid s determination of the osmotic pressure of the same species. The analytical evidence becomes still more consistent if it be assumed that the dog hemoglobin molecule contains 4 atoms of iron, 7 of sulfide sulfur, and 12 of sulfur. The molecular weights calculated on this basis from these t hree hemoglobin constituents are 66,476, 67,011, and 67,752.s As a result of studies of the oxygen-combining capacity of hemoglobin in the presence either of carbonic acid or of base, Adair has suggested that the molecule of hemoglobin may contain 4 atoms of iron and, therefore, have a molecular weight of 66,800. Finally Van Slyke (71) has determined the argininc, histidine, and lysine contents of ox hemoglobin, and Hanke and Kocssler (30) the histidine content of ox, horse, and cat hemoglobin. Only in the case of the arginine was the content of the amino acid low enough to yield a sufficiently high value for the weight of the protein containing 1 molecule, to be considered significant in the estimation of its molecular weight. The smallest weight of ox hemoglobin that can contain 1 molecule of arginine is 4,107 gm. Four times this value equals 16,428 gm., and eight times equals 32,856 gm., results which are in fair agrcemcnt with the molecular weight calculated above. Hemocyanin.-Since the copper and sulfur contents of the hemocyanins of various forms are different, it follows that their molecular weights must also be different. The difference between the hemocyanins of certain Cephalopoda and Arthropoda is particularly striking. Thus the hemocyanin of Octopus contains more copper than does that of Limulus, while the latter * That the true molecular weight of the hemoglobins not only of the dog, but also of the ox and of the horse, is either50,000 or G7,OOO will be shown in a subsequent communication. Ultrafiltration and dialyzing experiments have shown that hemoglobin is larger than serum albumin (7), and that these three hemoglobins have very similar molecular dimensions.

16 736 Physical Chemistry of Proteins. V contains nearly twice as much sulfur as the former. According to Henze (33) Octopus hemocyanin contains 0.38 per cent of copper. A calculation similar to that employed with the iron of hemoglobin leads to a molecular weight for this protein of 16,729. The sulfur content, 0.86 per cent, indicates that the smallest weight that can contain 1 atom of that element must be 3,729. Four times 3,729 leads to a much lower value for the minimal molecular weight than that estimated from its copper content. Five times 3,729 leads to a value that is too high. If these analytical values are assumed to be correct, the smallest Oclopus. Limulus. - - TABLE Minimal Molecular TVei$hts of the Hemocyanins. Method. Copper content (33). Sulfur (33). Copper ( 5). Arginine (71). Sulfur ( 5). Lysine (71). Histidine (71). II. per cent elm , , ,704 2,198 2,056 2,040 1, ,458 33,561 22,704 21,980 22,, ) ,813 molecular weight that can be attributed to this protein must be 33,500. For if it is assumed that this hemocyanin molecule contains 2 atoms of copper, its weight becomes 33,458; and if it contains 9 atoms of sulfur, it becomes 33,561. We may, therefore, take the minimal molecular weight of this protein as 33,500. A similar calculation has been made on the basis of Alsberg and Clark s analysis (5) of copper and of sulfur in the hemocyanin of Limulus, and is reported in Table II. The hi&line, arginine, and lysine contents of Limulus hemocyanin determined by Van Slyke (71) have also been used in this calculation. The minimal molecular weight of this substance is unquestionably different

17 Cohn, Hendry, and Prentiss 737 from that of the hemocyanm of Octopus, as Alsberg maintained, and is probably 22,700. For a summary of analyses and of containing weights of various hemocyanins, reference may be made to the article by Quagliariello (64). Egg Albumin.-At the time these investigations were undertaken, egg albumin was the only protein whose molecular weight had been adequately estimated from the osmotic pressure which TABLE Minimal Molecular Weight of Egg Albumin. Method. Osmotic pressure (70). Cystine content (31). I (43). Tryptophane (21). Histidine (60). Sulfide sulfur I (57). Aspartic acid I (60). Tyrosine (21). Lysine I (60). Arginine (60). Phenylalanine I (60). Proline (60). Sulfur I (57). Maximal base-combining capacity.6 III ,76C , , ,07c , ,05C 4.2 4, , , , , ,934 1,250-1 i 1 1, I,,, I - 34, , , , , , , , , , , , , ,750 it produces in the neighborhood of its isoelectric point. As a result of a painstaking series of investigations, in which corrections for membrane equilibria were employed, S. P. L. Sorensen and his collaborators (70) came to the conclusion that the molecular weight of egg albumin was approximately 34,000. Folin and Looney estimated the tryptophane and tyrosine contents of egg albumin as 1.23 and 4.2 per cent respectively. On the basis of the tryptophane content, the molecular weight of egg albumin must be at least 16,593. Twice this value, or 33,186,

18 738 Physical Chemistry of Proteins. V is in excellent agreement with the molecular weight calculated by Sorensen from osmotic pressure measurements. A molecule of egg albumin that contains 8 molecules of tyrosine would, by a similar calculation, have a molecular weight of 34,496. Any difference in the assumed number of tyrosine molecules leads to a dif?erence of over 4,000 in the calculated molecular weight. Therefore, this determination also confirms the estimate of the molecular weight of egg albumin. Egg albumin also contains cystine, but Folin and Looncy do not report a determination of the amount of this amino acid present. Harris (31) has estimated that egg albumin contains at least per cent of cystine, and more recently Jones, Gersdorff, and Moeller (43) have found 0.88 per cent, using Folin and Looney s calorimetric method. On this basis, the molecular weight would bc greater than 27,000, a result which cannot be considered as quantitatively satisfactory, but which nevertheless furnishes evidence of the size of this molecule. There is more sulfide sulfur in egg albumin, as in edestin and casein, than can be accounted for on the basis of its cystine content. Osborne (57) has determined both the sulfide sulfur and the total sulfur contents. There is too large an amount of total sulfur in egg albumin to render its determination of great value in minimal molecular weight calculations. If, however, one assumes that the egg albumin molecule contains 5 atoms of sulfide sulfur, its molecular weight becomes 32,660. The fact that a molecule of this size contains 1 molecule of cystinc or 2 of cysteine, and a prime number of sulfide sulfur and of sulfur atoms, makes the estimate of its minimal molecular weight independent of, but in excellent agreement with, Sorensen s osmotic pressure measurements. In 1909 Osborne, Jones, and Leavenworth (60) completed an analysis of the hydrolytic products of this protein. Their results for histidine, arginine, aspartic acid, phenylalanine, and proline, which have been included in Table III, all support the conclusion of Sorensen, that the molecular weight of egg albumin is approximately 34,000. The tryptophane, tyrosine, and sulfur determinations render 33,800 the most probable value. Casein.-The minimal molecular weight of casein also depends in large part upon its tryptophane content (21). Until recently the minimal molecular weight of casein has generally been assumed

19 Cohn, Hendry, and Prentiss 739 to be approximately 4,300. This estimate has been based on its phosphorus and on its sulfur contents. In a recent investigation, Cohn and Hendry (14) have determined, by the solubility method, that the equivalent combining capacity of casein for NaOH, in the neighborhood of its isoelectric point, lies between 2,006 and 2,166 gm. In another investigation, Cohn and Berggren (12) have determined, by means of electromotive force measurements, that TABLE IV. Minimal Molecular Weight Method. Tryptophane content (21). I (16). Histidine (59). I (30). Phosphorus ( 8). Sulfur I (29, 47). Phenylalanine I (22). Arginine S (59). Tyrosine (21). (20). Aspartic acid (1 3. Equivalent base-combining capacity (14). /%Hydroxyglutamic acid content (16). Ammonia I (59). Cystine I Sulfide sulfur of Casein.,er cent mn. 3, , , , , , , , , , , , , , ,253 12,006 12,408 10,922 13,116 12,660 12,765 13,710 13,516 12,556 12,984.2,600.2,424 12,696 (21) , ,080 (43) , ,384 (57) l,i52 3 )5,256 the maximal base-combining capacity of casein was 546 gm. Four times the equivalent combining weight at saturation yields 2,184 gm., a value which is in good agreement with the equivalent combining weight for base near the isoelectric point. The equivalent weight derived from solubi1it.y measurements is more accurate than that derived from electromotive force measurements. Six times the lower estimate derived from solubility

20 Physical Chemistry of Proteins. V measurements equals 12,576, and six times the higher equals 12,996. The molecular weight derived from Folin and Looney s tryptophanc determination is 13,253, and that derived from Dakin s gravimetric determination is 12,006. Six times the average of the limiting combining weights is 12,786, and of the tryptophane-containing weights is 12,629. Therefore, 12,800 may be taken as the minimal molecular weight of casein. Besides tryptophane, casein contains several other amino acids in such small amounts that they render still more certain this estimate of its minimal molecular weight. A casein molecule of this size would contain 2 molecules of histidine, 3 of phenylalanine and arginine, and 4 of tyrosine and aspartic acid. The minimal molecular weight calculated from each of these component parts of the casein molecule falls between 12,400 and 13,700. Two component parts of the casein molecule indicate that its true molecular weight must bc larger than, and some multiple of, 12,500. Osborne (57) long since determined the sulfide sulfur content of casein. A molecule of casein containing but 1 atom of sulfide sulfur must weigh 31,752 gm. Folin and Looney (21) find as little as 0.25 per cent of cystinc in casein, and Jones, Gersdorff, and Moeller (43), using the same method, have found almost precisely the same amount, 0.26 per cent. -4 molecule of casein containing 1 molecule of cystine, or 2 of cysteine, must therefore weigh approximately 96,000 gm. Such a molecule would contain 3 sulfide sulfur atoms, for three times 31,752 equals 95,256. The sulfide sulfur and the cystine contents of casein indicate that its minimal molecular weight must be far greater than 12,800, the value at which we arrived from t,he consideration of the other amino acids that casein contains and from its combining capacity. If we multiply this accurately known minimal molecular weight by 7, the result is, however, not in very good agreement with the molecular weight postulated from the sulfide sulfur or the cystine content. Eight times the minimal molecular weight leads to a result which is in no better agreement. If we proceed tentatively on the basis of these analytical results, allowing ourselves to be persuaded of their accuracy because of t,heir consistency, we must consider the molecular weight of casein as fifteen times 12,800, or 192,000. This estimate is precisely twice that demanded by

21 Cohn, Hendry, and Prentiss 741 the cystine, or four times that demanded by the cysteine, content of this protein. We have long hesitated to assume so great a molecular weight as 192,000 for any protcin.g It is impossible, however, to reconcile these different analytical results on the basis of a smaller molecule. Zein.-In considering the relation between the composition of zein and its acid and basic properties, we have recently had occasion to estimate its minimal molecular weight from a simultaneous consideration of its composition and its base-combining capacity. Cystine and histidine would seem to be the amino acids in which zein is poorest. If zcin contains 0.8 per cent of histidinc the molecular weight of zein cannot bc less than 1 3, There is some reason to believe that no very great change will occur in this estimate of the minimal molecular weight. For if the zcin molecule contains 2 molecules of arginine its weight would be 19,344; 3 molecules of P-hydroxyglutamic acid bring the molecular weight to 19,752; 3 of aspartic acid to 22,182; and 4 atoms of sulfur to 21,380. Although it is probable that all of these values will change as methods of separation and analysis improve, the very high known percentage composition of zein on the one hand, and the frequency with which a small number of assumed molecules leads to a molecular weight near 20,000 allows us to assume a figure of this order for the minimal molecular weight of zein with a fair degree of probability (13). Measurements of the base-combining capacity of zcin lead to an equivalent weight which is approximately one-sixth as great as the minimal molecular weight postulated on the basis of its histidine content. Two zein preparations were studied, one of which bound and the other mol of sodium hydroxide per gram (13). The equivalent weights of zein bound by 1 mol of sodium hydroxide were, therefore, 3,226 and 3,571 respectively. Six times the former leads to a minimal molecular weight of 19,356, and six times the latter combining weight. t o 21,426. These resuks must be considered in fair agreement with t.he minimal molecular weight estimated from analyses of zein. The true molecular weight of zein may be very much larger than 20,000. Folin and Looney have found only 0.5 per cent of cystine in zein hydrolysates by their calorimetric method. If this estimate is correct the molecular weight cannot be less than 48,040. Osborne had previously found that zein contained but a small amount of sulfide sulfur, and had accordingly predicted a high molecular weight. He found but per cent.of sulfide 9 See Cohn and Hendry (14), p, 549.

22 742 Physical Chemistry of Proteins. V sulfur. On the basis of this determination 15,127 gm. of zein would contain but 1 such atom. Three times this value leads to a molecular weight of 45,381, which is in good agreement with the molecular weight postulated on the basis of the cystine content. This coincidence lends a certain weight to both values, and also suggests that all of the sulfide sulfur in zein cannot represent cyst&e (13). I- T4nT.z v. Minimal Molecular Weight Zein. Method. Histidine content (61). Arginine (44). Aspartic acid (19). &Hydroxyglutamic acid (19). I Sulfur (57). Tyrosine (21). Rlaximal base-combining capacity (13). er cent Qrn. 1.8,91i 9,56t 7,391 6,522 5,34i 3,234 3,4Ol Cystine content (43). 0.85!8,25! Histidinc < (61). 0.82!8,91! Sulfide sulfur I (57). 0.21: 15,12: Arginine (44) ,56t Aspartic acid (19) ,39< Sulfur (57) ,34! I Cystine Histidine Sulfide sulfur I Arginine Aspartic acid Sulfur (21). 0.5 (61) L 3 K!3,04( 18,91! (57). 0.21:!I 15,12 (44) ,561 (19) ,391 (57) ,34! 1 18, , , , , , , , , , , , , , , , , , ,210 Since these calculations were made, Jones, Gersdorff, and Moeller (43) have reported a cystine content in zein of 0.85 per cent. If this higher result is correct the cystine-containing weight is 28,259. Such a molecule would contain 2, rather than 3, sulfide sulfur atoms. In zein, as in cascin, the cystine content suggests a higher molecular weight than does any other amino acid. For neither

23 Cohn, Hendry, and Prentiss 743 of the minimal molecular weights calculated from these different analyses of cystine is an integral multiple of 19,400. Jones, Gersdorff, and Moeller s cystine determination leads to a minimal molecular weight of approximately 58,000; Folin and Looney s to one of 96,000. Both would contain 2 cystine or 4 cysteine molecules, whiie the former would contain 3, the latter 5, histidine molecules. The physicochemical and analytical data have been calculated and tabulated on both bases. The weights of zein containing sulfur and aspartic acid, which in the first computation led to slightly abnormal values, conform admirably to a minimal molecular weight of approximately 58,000 or 96,000. Nevertheless the discrepancy between the cystine estimates is such that it seems preferable, pending its redetermination, to consider 19,400 as the minimal molecular weight of zein. G&ad&.-The minimal molecular weight of gliadin can be estimated with great precision both by analytical and by physicochemical means. The very small number of free acid and free basic groups in the prolamins renders electromotive force measurements of their equivalent weights particularly valuable in the estimation of their minimal molecular weights. The tryptophane content of gliadin has been determined both gravimetrically and calorimetrically. Abderhalden and Samuely (2) found approximately 1 per cent of tryptophane in gliadin, Folin and Looney (21) estimated that a slightly larger amount was present, namely 1.14 per cent, and Cross and Swain (15) found 1.11, 1.19, 1.03, and 1.13 per cent in preparations from different flours. May and Rose (52), using a comparative colorimetric method based upon the amount of tryptophane in casein, estimated that gliadin contained 1.05 per cent. Since casein probably contains nearer 1.6 than 1.5 per cent, their results are probably low by about 6 per cent. These four different investigations leave little doubt that gliadin contains between 1.0 and 1.14 per cent of tryptophane. The minimal molecular weight of gliadin Gahhted from the lowest value is 20,410, and from 1.14 per cent is 17,904.lO 10 If Folin and Looney s tryptophane and cystine estimates are both correct, the molecular weight of gliadin cannot be less than 72,000. The sulfur content of gliadin leads to a containing weight which also suggests 72,000 rather than 20,700, but the lysine content would demand a molecule of twice this size.

24 744 Physical Chemistry of Proteins. V The lysine content of gliadin is also very small. Van Slyke s most recently published determination of this quantity, 0.69 per cent (71), leads to a minimal molecular weight of 21,173. The tryptophane and lysine contents of gliadin, therefore, suggest a minimal molecular weight in the neighborhood of 20,000. A number of other amino acids are present in gliadin in very TABLE Minimal Molecular Weight of Gliadin. Method. Tryptophane content (21). I ( 2). Lysine (71). Cystine I (21). &Hydroxyglutamic acid (17). Sulfide sulfur (57). Histidine I (71). Arginine (71). Tyrosine (21). Maximal base-combining capacity (25). < Sulfur content (57; VI. er cent Qrn..7,901 to, 41(!1,17Z 0,35: 6,79( 5,181 4,63f 5,544 5,174 3,33: 3,441 3,12: %3 3 2.P 2 o- 1 17, , , , , , , , , , ,688 7 ~_ 21,861 small amount. Thus, Folin and Looney s cystine determination (21) leads to a minimal molecular weight of 10,353, and Jones, Gersdorff, and Moeller s (43) lower determinations, to slightly higher values. On the basis of Folin and Looney s estimate, 2 molecules of cystine, or 4 of cysteine lead to a minimal molecular weight of 20,706; 3 of P-hydroxyglutamic acid, to 20,388; and 11 Cross and Swain have used Van Slyke s method in estimating the diamino acids. Whereas the sum of the average results for all these acids is equal to the sum of the histidine, arginine, and lysine determinations of Van Slyke, the variations between their different analyses were so great as to preclude their use in the calculation of containing weights. Their tyrosine values are doubtless a little high owing to the residual color of the reagent (15), and are therefore not considered.