THE CHEMICAL NEWS AND JOURNAL OF PHYSICAL SCIENCE Edited by Established (WITH WHICH IS INCORPORATED THE "CHEMICAL GAZETTE"). [ in the Year 1859. Published Weekly. Annual Subscription. free by post 1 Entered at the New York Post Office as Second Class Mail Matter. Transmissible through the Post-United Kingdom, at Newspaper rate; Canada and Newfoundland, at Magazine rate. Jan. 9, 1920 WHATMAN Grade FILTER PAPERS Note a few of our later specialities: For use with No. 42-"ASHLESS." Double Acid-washed and extremely close of texture. No. 50-Hardened by treatment with Nitric Acid. Very tough, it will resist great pressure, and SOLE MANUFACTURERS: W. & R. BALSTON, LTD., MEDWAY MILL, MAIDSTONE, KENT. FIND OUT ALL ABOUT OUR COMPLETE RANGE FROM OUR REVISED BOOKLET AND PRICE LIST. In case of difficulty in obtaining Free Samples, write the Sole Mill Representatives- CH3 CH.CHNH2.COOH CH3 CH2 NOTES ON THE EVOLUTION OF PROTEINS. By E. L. KENNAWAY, M.D., D.Sc., Middlesex Hospital, London. UNTIL recent years evolution has been studied almost exclusively by morphological methods. The most detailed pictures of evolution which we possess, namely, those showing the gradual construction of the modern horse and elephant, have been brought together solely by the inspection of bones; this is unavoidable in the study of extinct forms, since the materials most valuable to the biochemist are not preserved in fossils. The application of experimental methods, as in the study of genetics, for instance, has of course yielded results of the utmost value, but the mode of observation is still chiefly anatomical; the subject of biochemical evolution is still to a large extent untouched. Since organisms which are, anatomically, very simple still exist side by side with others which are very complex, one would think it possible by the study of the composition of both types to investigate the chemical basis of the transition from the one to the other. One may consider here the question of the evolution of proteins. During the past seventy years an immense amount of labour has been expended in attempting t trace the course of the evolution of animals; yet is it possible at the present day to give an account of the steps by which any mammalian protein, for instance the caseinogen of milk, has been produced? Every protein which has been analysed is found to be composed of amino-acids. The amino acids which have been established as occurring commonly in proteins are seventeen in number (see Note); the composition of these is given in Table I. 5. Isoleucine .. 12. Paenylalanine C6H5.CH2.CHNH2.COOH 14. Tryptophane. C6H4 (NOTE.-Improved analytical methods will no doubt show that the actual number is greater, but this scarcely affects the considerations put forward in this paper. A new amino-acid (3-hydroxyglutamic acid) was discovered in caseinogen quite recently (1); it is omitted from the table because it has not yet been sought in other proteins. Normal leucine is also omitted because there are as yet very few data as to its distribution. Several others, in addition to the seventeen, have been described (Plimmer, "Chemical Constitution of the Proteins," 3rd Ed., Part I., p. 5). Of these, some (caseinic acid, caseanic acid, hydroxyamino-succinic acid, hydroxyamino-suberic acid, dihydr- 15. Proline .. oxy-diamino-suberic acid, hydroxy-diamino-sebacic acid) are regarded by Plimmer as mixtures, and some others (diamino-trioxydodecanic acid, oxytryptophane) are probably secondary products produced in the course of analysis. The occurrence of amino-butyric acid is disputed. The remainder (dihydroxy-phenylalanine, di-iodotyrosine) are so closely related to some which appear in the table that, from the present point of view, they may be disregarded. The deficiency in the yield of pure amino-acids by Fischer's method, the yield amounting in many cases to not more than 50 per cent of the weight of the original protein, does not in itself offer any strong evidence of the existence of undetected amino-acids. The sources of loss, especially in the final isolation and purification of the individual amino-acids, are very serious, and it is noteworthy that the best yields-80 to 90 per cent-have been obtained from those proteins in which a large fraction of the molecule is made up of but one or two amino-acidse.g., wheat-gliadin with 35 per cent of glutamic acid, silk fibroin with 60 per cent of glycine+alanine; in such cases the summation of losses incurred in the isolation of a number of different acids is avoided). 16. Oxyproline 17. Histidine C-CH2.CHNH2.COOH CH NH It is at once evident on inspection of Table I. that the organisms which synthesise amino acids make a most extraordinary selection. The object of this paper is to direct to this point more attention than it seems to receive. The selection is remarkable in two ways, namely, (1) it is very limited as regards number, but (2) it is very diverse as regards structure. The total possible amino-acids of complexity lying within the range exhibited by the above ten classes must amount to a very large number. On what basis is this number reduced to seventeen or so? What is the meaning of the curious assortment of ring compounds? Why should two such compounds as, for instance, glutamic acid and tryptophane be chosen? The selection might have been much less, or much more, diverse; as it stands it has a curiously arbitrary appearance. It may, of course, be said that the organism reaches this series of amino-acids, not by any process of selection from a greater number, but through its inability to synthesise any others. This might be so, though it scarcely renders the matter any less remarkable. But in view of the great synthetic capacity of plants, as shown by the structure of alkaloids, for instance, it is difficult to believe that other amino-acids could not be synthesised if they were required. Again, it may be said that the existing series of amino-acids has been selected because these, in combination together in varying patterns and numbers, give products which have the properties required of proteins. This is no doubt the case, but it is little more than a paraphase of what is obvious. To attain to any satisfactory knowledge in this direction one requires to know, firstly, what would be the characters of protein" made from an altogether different set of amino-acids, and, secondly, by what process of trial and error the organism succeeded in making the most suitable protein from the most suitable materials. In seeking for information on the evolution of proteins the higher animals at any rate may be disregarded, since they appear under normal conditions to obtain their aminoacids, directly or indirectly, from plants; the limited powers of synthesis which they have been shown to possess are alluded to below. We have so little knowledge of the metabolism of the invertebrates that it is unsafe to assume anything as to their capacities in this respect. Thus, Loeb (2) has shown that the larva of the banana-fly can grow upon a medium containing no nitrogenous substance other than ammonium tartrate; the experiments did not decide whether this synthesis of protein was carried out by bacteria introduced with the eggs or not. The proteins of higher plants, the wheat-plant, for instance, are known to contain all the amino-acids. The problem of the evolutionary chemistry of proteins, in so far as it is accessible at the present day, resolves itself then to this: do the simplest plants (e.g., bacteria, yeast) contain all the amino-acids present in the higher plants? If not, in what plants do the others appear? Such information as is available no this question is given by Plimmer in a table in the latest edition of "The Chemical Constitution of the Proteins " (Part I., p. 127). This shows that various workers have analysed the proteins of five species of bacteria, of yeast, and of a mould (Aspergillus niger); and the table includes analyses of a protozoon (Noctiluca).* Table II. is adapted from that given by Plimmer; the composition of caseinogen, a protein peculiar to mammals, has been in cluded for the sake of comparison. The analyses of yeast are those of three different workerst; with regard to four of the amino-acids they are not in agreement. Processes for the isolation of the protein were carried out in the cases of the first four bacteria in the table, and of Neuberg's (9) yeast; Aspergillus niger was extracted with alcohol and ether before analysis; in the remainder (Azotobacter and two yeasts) no separation of the nonprotein constituents was attempted, so that unfortunately it is uncertain whether the amino-acids found in them are actually combined as protein. Analyses of the proteins of other protozoa would be of much interect; mycetozoa, such as Badhamia should provide a sufficient supply of material. Neuberg's (9) paper has been available only in the form of an abstract. The table shows that the five bacteria, yeast, and the mould, taken together. contain all the seventeen aminoacids with the exception of oxyproline, the presence or absence of which was not established, and of serine and cystine, the presence of which was doubtful (in yeast). With regard to oxy proline, "only in a few cases has this compound been isolated from the products of hydrolysis of proteins, since its separation is extremely laborious" (Plimmer). It is recorded as present in 8 only of 157 analyses of proteins given by Plimmer. Moreover, proline is present in five of the seven micro-organisms, so that from the present point of view the presence or absence of its hydroxy compound is not of importance. The absence of cystine from the bacterial proteins, which was established by the negative result of a test for sulphur, is noteworthy; this compound is present in numerous proteins of the higher animals and plants.* As the synthetic powers of these organisms are of especial interest it is unfortunate that only two of them were grown upon media of known composition, namely, Aspergillus niger (on MgSO4, KH2PO, KCl, FeSO4, KNO3, cane sugar) and Mycobacterium (on MgSO4, K2HPO4, NaCl, glycerin, ammonium lactate, asparagin). The other bacteria were all grown upon agar or bouilion media, and no doubt some of them could not have dispensed with certain materials, perhaps of the character of vitamines, contained in these complex mixtures. Pringsbeim (17) used ordinary "Presshefe," and Meisenheimer (10) yeast which seems not to have been grown on special media; no details as to Neuberg's yeast (9) are accessible. However, one must take these scanty results as all that are at present available. They show that the simplest organisms now existing do not contain a series of aminoacids any more primitive than that present in the higher organisms, except perhaps as regards the inclusion of cystine. One may suppose that the present apparently stereotyped series of utilisable amino-acids represents the stable outcome of a struggle long ago among simple organisms in which those which made a less suitable choice were beaten, and have passed away leaving no trace. We cannot know the biochemistry of the first organisms which appeared upon the earth; the experi ments and discarded compounds of that time are lost. The selection of amino acids must have taken place at an immensely remote period, for the earliest records which we have of the forms of life on the earth do not show us organisms which have any appearance of noteworthy difference in chemical composition from those which exist at the present day. Thus one of the earliest fossils, the brachiopod Lingula of the Cambrian period, is very closely related to a species now living. Structures regarded as fossil Algæ very similar to recent species have been found in still older rocks. The doctrine of natural selection gives the impression that evolution proceeds throughout in a very gradual manner. But at the time wben the aminoacids were first being produced and tested organic evolution must have proceeded very distinctly per saltum as each new compound was synthesised; natural selection would then act slowly and surely upon the organisms which made one or another choice, and thus the present series of amino-acids was delimited. An obvious instance of the apparently "arbitrary" nature of the selection is the avoidance of the four-carbon acid, while the 2-, 3-, 5-, and 6-carbon acids are utilised, the 3 carbon acid moreover appearing in five forms (alanine, phenyl-alanine, tyrosine, tryptophane, and histidine) and the 6-carbon acid in four (leucine, iso-leucine, normal leucine, and lysine). Some of the amino-acids There is some indirect evidence that cystine is present in the protein of yeast, since this can serve as the sole protein food of rats (25). Walcott, quoted by Adami, "Medical Contributions to the Study of Evolution," London, 1918 p. 17. The geological history of the bacteria is of course an uncertain matter, depending chiefly upon the inference that various rocks were deposited by bacterial action; the literature on this subject is referred to by Adami (oc. cit.). TABLE II. (Blank spaces indicate that the presence or absence of the amino-acid was not determined). may have been chosen because of their suitability, not only for the formation of proteins, but for the production of other compounds as well. Thus arginine and histidine may serve as sources of purines (12); proline is closely related to a group present in chlorophyll and bæmoglobin; and tyrosine to adrenalin and perhaps some pigments. One does not of course suggest that the earliest organisms provided for the future formation of substances such as chlorophyll and adrenalin; but the various chemical possibilities of these amino-acids may be utilised by the simplest plants in their metabolism in ways which we do not know of. Pringsheim (17) has considered this point in the case of yeast. There is obviously much to be learned as to the experimental methods by which organisms arrive at the production of suitable substances, for any given new compound cannot be brought into existence gradually. The final, or rather the present, product is in many cases a highly successful one (e.g., the superiority of adrenalin to many allied compounds (13); the great potency of snake venom), but the preceding stages are effaced by natural selection. Very little seems to be known also of the method of synthesis of the amino-acids in plants; possibly the plant makes from dextrose those members of the series which the mammal can convert into dextrose. It has long been taught that animals are dependent ultimately upon plants for their supply of protein. Knoop '15 and Embden and Schmitz (17) and their fellow workers (18, 19) showed however, in some experiments which attracted a great deal of attention at the time (1910), that mammals have some power of synthesising amino-acids. But surely these experiments, striking as they are, reveal chiefly the feebleness of this synthetic power in animals; they seem rather to emphasise how completely parasitic upon the plant the animal has become. It was shown that the mammal, when supplied with certain a-keto-acids, could convert them into the corresponding a-amino-acids whether these were of natural occurrence (alanin, phenyl-alanın, tyrosin, a-amino caproic acid) or not (a-amino-butyric acid, v-phenyl-a-aminobutyric acid). But this insertion of an NH2 group is no great synthetic achievement. Firstly, as Knoop points out, this change in keto acids may occur in vitro simply in the presence of ammonium carbonate. Secondly, there was admittedly nothing in the experiments to show that the mammal could itself provide the essential molecule to which the NH2 group was to be attached, except the very simple one (that of pyruvic or lactic acid) required for the production of alanin. It is known further that mammals can produce the simplest amino-acid glycin; but whether by a synthetic process or otherwise is not clear (20). Tyrosine can be formed in the liver under experimental conditions from phenyl-alanine (14), and hence appears not to be an essential constituent of diet (15). Whether phenyl-alanine can be synthesised has not yet been shown. This appears to be all the evidence + + + O T 1 |