October, 1924 INDUSTRIAL A N D ENGINEERING CHEMISTRY 1029

Recent Advances in Protein Chemistry‘ By Hubert Bradford Vickery

CONNECTICUT AGRICULTURAL EXPERIMENT STATION, NEW HAVEN, CONN.

ROM the standpoint of the organic chemist the most interesting and a t the same time the most fundamental F problem of protein chemistry is that of protein con-

stitution. A knowledge of constitution, however, is de- pendent upon a knowledge of composition, and the compo- sition of proteins is a problem that is still far from solution. Proteins, when boiled with acid, are resolved into a mixture of a-amino acids, of which seventeen are now definitely known. The complete separation of these amphoteric substances was practically a hopeless undertaking until Fischer in 19Ol1, * introduced the method of fractional distillation of the ethyl esters under very low pressure. Since then the knowledge of the amino acid make-up of proteins has progressed enor- mously, so that by 1918 the composition of from 65 to 85 per cent of the molecule of a number of proteins was accurately known. The technical difficulties of this method were so great, however, that a higher summation of the individual amino acids could hardly be expected until a more precise knowledge of their properties had been obtained.

In 1914, Foreman2 showed that the calcium salts of di- basic amino acids could be precipitated from the hydrolysis mixture by means of alcohol. This method led directly to the discovery of oxyglutaminic acid in casein by Dakin3 in 1918. This amino acid has since been obtained from several other proteins and is perhaps quite widely dis t r ib~ted.~j~,6 At the time he announced the discovery of oxyglutaminic acid, 1)akin showed that the mono-amino acids could be separated as a group from the hydrolysis mixture, by Iong- continued extraction with butyl alcohol, in a form eminently suitable for esterification and subsequent separation by Fisch- er’s method. Dakin’s procedure is probably the most im- portant advance in the technic of protein analysis of recent years, and has enabled him to increase the summation of amino acids in gelatin to 91.3 per cent17 and in zein, the most abundant protein in the maize kernel, to 101.5 per cent.8 These are unprecedented figures. I t seems highly improbable that any appreciable amount of a hitherto unknown amino acid can occur in zein, and the day does not seem far distant when we shall know the composition of the whole of the molecule of at least one protein.

Although the method introduced by Kossel and Kutscher for estimating the basic amino acids in proteins leaves little to be desired on the score of accuracy, there is room for much improvement in convenience. An observation of Foster and S ~ h r n i d t ~ , ~ ~ is very promising. They found that the basic amino acids arginine, histidine, and lysine migrate to the cathode when a hydrolysis mixture is electrolyzed under proper conditions of acidity, and may be obtained free from mono- and dibasic amino acids by a second treatment. More- over, histidine can be separated from the other two bases by similar means. If this procedure can be placed upon a strictly quantitative basis, one of the most laborious and time-consuming operations in protein analysis will be elim- inated.

Colorimetric methods to estimate tryptophan, cystine, and tyrosine have been developed by Folin and Looneyll to a very hrgh degree of accuracy, with the result that the trypto- phan content of a number of proteins is now quite well estab-

1 Rt>ceived July 8, 1924. * Numbers in text refer to bibliography a t end of article.

lished. When it is recalled that this amino acid is essential in nutrition,lz the importance of this knowledge is evident. A colorimetric method to estimate histidine has been pro- posed by Koessler and Hanke,13 which also appears to give very reliable results.

G0rtnerl~,~5>~6 and his collaborators have studied the humin which forms when a tryptophan-containing protein is boiled with strong acids. They have collected evidence which strongly supports the view that the humin is formed by a condensation of tryptophan, or at any rate, the indole nucleus of tryptophan, with an unknown aldehydic constituent of the protein molecule.

The present tendency to utilize organic chemical reagents to make separations which cannot be made with inorganic reagents is well illustrated by the new method of Kossel and Grossl7 to isolate arginine. They have found that 2,4- dinitro-7-naphtholsulfonic acid precipitates arginine quanti- tatively from an acid hydrolysis mixture. Although the method has been used in only one other laboratory,18 it seems to be extremely valuable and suggests the possibility that other reagents may be discovered which will render the isolation of amino acids less difficult.

PROBLEMS FOR STUDY

At the present time there are several well-defined needs in the field of analytical protein chemistry.

In the first place a comprehensive study of each amino acid would be very useful. We need detailed and accurate in- formation regarding physical properties, solubilities of various salts and other derivatives, and knowledge of new compounds which might serve for purposes of isolation and estimation. Although much is already known, the field is only beginning to be opened.

Work of this kind is particularly necessary for the oxy- amino acids, serine and oxyproline. The latter has been isolated only a few times, and its separation from glycine is a t present extremely difficult.

The peptide anhydrides which are found, together with proline, in the butyl alcohol solution obtained in Dakin’s procedure, have not received much attention. They inter- fere seriously with the indirect estimation of this amino acid by the Van Slyke amino-nitrogen method. Some nieans to separate proline from them would be extremely welcome. Abderhalden has used organic solvents to extract peptide anhydrides from hydrolysis mixtures. It is possible that they could be used to make this separation, but the solubility of proline in such solvents is not well known.

Finally, a method is needed to estimate, and if possible to precipitate,.phenylalanine. It does not seem impossible that the properties of the benzene ring in this amino acid may be utilized to convert it either into an insoluble substance or into a derivative suitable for colorimetric estimation. The chief difficulty to be foreseen is the presence of tyrosine, which would probably share in any such chemical conversions.

NEW VIEW OF YATURE OF PROTEINS

Osborne long ago showedlg that the protein edestin had distinct basic properties and could unite with one or with two equivalents of acid, forming definite crystalline compounds.

1030 1

INDUSTRIAL A N D ENG

Moreover, numerous other proteins can be prepared in crys- talline form and exhibit many of the properties of pure sub- stances. Nevertheless, chemists have often failed to recognize the definiteness of the physical and chemical properties of pure protein preparations and have been inclined to classify them as colloids, attributing their capacity for uniting with acids and bases to adsorption. This point of view is now under- going a marked change. Loeb20 has recently demonstrated that many properties of proteins are more readily explained by the laws of classical chemistry than by the laws of colloids. Cohn12* using strict physico-chemical methods, has shown that proteins have a perfectly definite solubility in water, which may be utilized as a means of identification, and has investigated the solubility of casein in sodium hydroxide22 from the standpoint of the law of mass action. He has found that the equivalent weight of casein is very close to 2100. The minimum molecular weight can be calculated from the proportions of sulfur, phosphorus, and of certain easily de- termined amino acids in the protein, Cohn shows that there is considerable probability that the minimum molecular weight of casein is of the order of 12,600, since several in- dependent sets of data lead to the same conclusion.

One of the leaders of thought in this more logical view of the nature of proteins has been L. J. Henderson, who has pointed out25 that in the presence of acids or of alkalies pro- teins behave as if they possess a number of acid and basic radicals of varying strengths. I n an acid solution some, or all, of the basic radicals are in combination depending on the acidity; in alkaline solution some, or all, of the acidic radicals are in combination depending on the alkalinity. At a certain definite hydrogen-ion concentration the amount of protein in combination with either acid or alkali is at a minimum, which may be zero. At some point the ionizations of the protein through its acid and basic radicals must become equal and at this, which may be termed the isoelectric point, properties which are chiefly due to the presence of protein ions will pass through minima-for example, solubility, swelling, and viscosity. On the other hand, properties chiefly due to the presence of undissociated molecules will pass through maxima-for example, flocculating power. Theoretical ti- tration curves for a hypothetical protein possessing acid and basic radicals of definite dissociation constants can be con- structed which simulate very closely the titration curves actually obtained from proteins.

Salt formation in the strictest sense of the term, with all the qualitative and quantitative implications, is entirely adequate to explain the behavior of proteins with small concentrations of acids and alkalies. With greater concen- trations nothing is known which is incompatible with this view, although under extreme conditions unexplained ir- reversible changes occur which result in the so-called “de- naturing” of the protein.

An excellent example of the rigid application of physical chemistry to proteins is furnished by recent work of Van Slyke and his collaborators on the chemistry of hemoglobin.24 They find that the increase in base binding power that occurs when reduced hemoglobin is oxygenated a t varying hydrogen- ion concentration follows a curve which is quantitatively consistent with Henderson’s view that combination with oxygen increases the dissociation constant of one acid hydro- gen in the protein molecule. The experimental curves are accurately expressed by equations derived from the mass law.

In recent years we have learned, therefore, to look upon proteins, not as mysterious colloidal complexes, but as chem- ical individuals of, in certain cases, quite well-known com- position, with properties which conform to the laws of clas- sical chemistry.

VNEERING CHEMISTRY Vol. 16, No. 10

It should be pointed out, however, that proteins can, and often do, occur in colloidal form. The colloid chemists have quite rightly claimed certain of the properties of these sub- stances for their field; but they have often been misled by a lack of appreciation of the capacity of proteins to enter into true chemical combination with other substances. More- over, the difficulties encountered by many are readily ex- plained by a lack of purity of their protein preparations. Chemists have frequently carried out elaborate research with purified reagents and highly accurate physical apparatus upon grossly impure material.

PRACTICAL APPLICATIONS The study of proteins has innumerable practical applica-

tions. The recent advances in the knowledge of the food value of proteins are directly traceable to the analytica1 studies made from ten to fifteen years ago. For example, it is now recognized that the protein inadequacies of corn in animal nutrition can be wholly supplemented by skim milk or meat scrap.

An animal may be sensitized by a dose of a milligram or less of protein injected into the peritoneal cavity. If the same pro- tein is later injected in larger dose, severe reactions, which are often fatal, occur. This phenomenon places the whole problem of immunity and hypersensitivity in the field of pro- tein chemistry. As is well known, hay fever is now success- fully treated from this standpoint, and there is much evidence that the toxins and antitoxins of bacterial disease are protein in nature. Evidence has also been obtained that insulin is a protein.25

The physiological activity of proteins is startling.

CONCLUSION

Chemists have every reason for gratification at thewre- markable achievements in the field of protein chemistry in recent years. A much clearer comprehension of the problems has been attained, and the ground is well prepared for future research, which will undoubtedly yield results of the great- est importance.

BIBLIOGRAPHY 1-Fischer, Z . physiol. Chem., 33, 151 (1901). 2-Foreman, Biochem. J . , 8, 463 (1914). a-Dakin, Ibid., 12, 290 (1918). 4--Dakin, Ibzd., 13, 398 (1919). 5-Dakin, 2. physiol. Chem., 130, 159 (1923). 6-Jones and Johns, J . Biol. Chem., 48, 347 (1921). 7-Dakin, Ibid , 44, 499 (1920). 8-Kossel and Kutscher, Z. physiol. Chem., 31, 165 (1900). 9-Foster and Schmidt, Proc. SOC. Exptl . Bid. Med., 19, 348 (1922).

10-Foster and Schmidt, J . Biol. Chem., 66, 545 (1923). 11-Folin and Looney, Ibid., 61, 421 (1922). 12-Osborne and Mendel, Ibid., 17, 325 (1914). 13-Koessler and Hanke, Ibid., 43, 621 (1920). 14-Gortner and Blish, J . Am. Chem. Soc., 37, 1630 (1915). 15-Gortner and Holm, Ibid., 39, 2477 (1917). 16-Gortner and Norris, Ibid., 45, 550 (1923). 17-Kossel and Gross, Z . physiol. Chem., 136, 167 (1924). 18-Karrer, el al., Ibzd., 135, 129 (1924). lg-osborne, J . A m . Chem. Soc., 24, 39 (1902). 20-Loeb, “Proteins and the Theory of Colloidal Behavior,” New York,

21-Cohn, J . Gen. Physiol , 4, 697 (1922). 22-Cohn, Ibid., 6, 521 (1923). 23-Henderson, “The Vegetable Proteins,” by Osborne, Chap. V,

24--Hastings, Van Slyke, Neil, Heidelberger, and Harington, J . Bid.

25--Somogyi, Doisy, and Shaffer, Ibid., 60, 31 (1924).

1922.

London, 1924.

Chem., 60, 89 (1924).

The College of the Pacific has moved from San Jose to Stock- ton, Calif., where seven new buildings are being completed a t a cost of $750,000, The science building will be wel1,equipped for physics, chemistry, and biology.