of the yeast cell, so that, even when the cell membrane has become warped by moisture and the other portions of the cell contents have disappeared from view, the granules remain alone, in the form of free globules (§ 8) as fine as dust, which swim in the nutrient medium, and occasionally render the latter turbid. Probably it is in this sense that we should regard the observations which J. WORTMANN (XII.) had occasion to make in connection with wines that had turned turbid during storage in bottle.

CHAPTER XLVIII.

CHEMISTRY OF THE YEAST CELL.

§ 252.-Chemistry of the Yeast Cell Nucleus.

RECENT investigations have shown the probability that, in place of being in a free state in the cell, the albuminoids present form part of more highly constituted bodies termed proteids. At a given moment these bodies are split up into the albuminous nucleus on the one hand, and the adherent lateral chains on the other. These latter have been termed prosthetic groups by A. KOSSEL (V.), and explained as the real means by which the vital activity of the cell is enabled to act. Of the groups into which the proteids themselves have been classified, one, namely that of the nucleins, is of particular importance, since, as the name itself implies, it furnishes important structural material for the nucleus. A little fuller information on this point will probably be not unwelcome to the reader, and for this reason a few remarks concerning certain conclusions of a practical nature will now be given.

In 1869 F. MIESCHER (I.) isolated, from the nuclei of pus cells, a nitrogenous constituent to which he gave the name nuclein. This body differed from other proteids, both as regards its percentage of phosphorus and by its power of withstanding pepsine. The occurrence of such nuclein in yeast cells was then discovered by F. HOPPE-SEYLER (IV.). After doubt had been cast by NÆGELI and LOEW (II.) on the accuracy of these observations, A. KoSSEL (III.) succeeded in obtaining considerable quantities of fairly pure nuclein from pressed yeast. For this purpose the yeast, stirred to a pulp, was left for several hours under water, the latter being renewed once or twice in the interim. The yeast sediment was then introduced into dilute caustic soda, which extracted the nuclein. As, however, the latter was at the same time gradually attacked by the solvent, it was found desirable to place the mixture at once on a number of filters and to allow the filtrate to drop into dilute hydrochloric acid, which re-precipitated the dissolved nuclein. The precipitates were then united on a filter, washed with dilute hydrochloric acid and alcohol, and repeatedly extracted with the same, thus furnishing a product which, when dried under the

air-pump, formed a white and fairly pure preparation. Analogous substances have been separated from the most divergent animal and vegetable organs, and classified under the collective term nucleins.

The first investigations concerning the percentage proportion of nuclein to the total nitrogenous constituents of yeast were performed by A. STUTZER (I.). According to this worker, the dried residue obtained from beer yeast by several days' cold extraction with 95 per cent. alcohol followed by drying over sulphuric acid, contained 8.65 per cent. of total nitrogen, of which 2.26 per cent. (or more than a quarter) was in the form of nuclein. Still richer in this respect was the thallus of a mould fungus of undetermined species, which settled from the air into a nutrient solution containing tartaric acid; since, out of 3.78 per cent. of total nitrogen in the dry residue, 1.54 per cent. (or nearly 41 per cent. of the whole) was present as nuclein.

It is to the labours of A. Kossel and his pupils that we are mainly indebted for information on the chemical constitution of nuclein in general, and of yeast nuclein in particular. Their discoveries furnished the basis for KOSSEL's (II.) classification of the nucleins into two groups. The first includes substances, which, so far as is known, do not occur in cell nuclei, and which were named paranucleins by Kossel, or pseudonucleins by HAMMARSTEN (III.). When decomposed by dilute acids they yield only phosphoric acid and albumin. On the other hand, under the same treatment, the true nucleins, which alone form the subject of the following lines, furnish, in addition, basic substances to which the name nuclein bases has been given.

An important elucidation of the constitution of nuclein was first presented by R. ALTMANN (I.) by the discovery that these proteids are decomposed by dilute alkalis into albumin and a nitrogenous acid, rich in phosphorus, which has received the name of nucleic acid. This therefore is the prosthetic side chain in the nuclein complex; and it generally receives an additional appellation indicative of the origin of the nuclein in question. All the varieties of nucleic acid exhibit the characteristic behaviour, when heated with dilute acids, that they suffer decomposition into one or other of the nuclein bases described below. The molecule of nucleic acid is composed of a large number of atoms. Those of phosphorus and nitrogen stand in the ratio : 3 in nearly all the instances known. So far, only two exceptions to this rule have been observed. One of them is the guanylic acid, isolated from the nuclein proteid of the pancreas of the ox and more closely examined by I. BANGS (I.), the molecule of which acid contains 5 atoms of nitrogen to each atom of phosphorus. The other is a nucleic acid isolated from the embryo of wheat by TH. B. OSBORNE and G. F. CAMPBELL (I.), the ratio P: N being in this case 1 : 4.

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As regards the composition of the albuminoids resulting from the decomposition of the nucleins, less is known than of the nucleic acids with which they are combined. A. KoSSEL (III.) was the first to investigate yeast nuclein, and he found that the albuminoid substance obtainable therefrom offered greater resistance to the digestive enzymes (pepsin and trypsin) than the yeast nuclein itself, since about 66 per cent. of the latter, but only 3 per cent. of the former could be brought into solution within twelve hours in the course of a comparative test.

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According to A. KOSSEL (II.), the yeast nucleic acid, first prepared by Altmann, has the formula CH2N6 P2014 or C25 H36 Ng P3O.22. The same authority (V.) states that it is decomposed by alkalis into carbohydrates and an acid, namely plasmic acid, rich in phosphorus and in nitrogen. The formula (C15H2 Ne PO30) ascribed to this latter acid by Kossel is apparently only approximately correct, since no notice was taken of the iron content (p. 47) subsequently found therein by A. ASCOLI (I.). Under the influence of boiling dilute mineral acids, plasmic acid furnishes nuclein bases, together with phosphoric acid and a nitrogenous organic substance which has not yet been more closely examined. A patent has been taken out by K. SCHWICKERATH (I.) for a process of purifying crude nucleic acid from adherent brown mucinous substances, in view of the preparation of yeast nucleic acid on a large scale. In this connection, attention should also be directed to the German Patent, No. 107,734, granted to the Elberfeld Farbenfabriken.

The carbohydrates furnished by the action of boiling dilute mineral acids on yeast nucleic acid (though not from plasmic acid), reduce Fehling's solution, and, according to KOSSEL (V.), probably consist of a mixture of glucose and one of the pentoses. LIEBERMANN and BITTO (I.) regarded them as an adherent contaminating admixture, of the character of the so-called yeast gum. Similar carbohydrates have also been isolated from various nucleins of animal origin by A. KOSSEL and A. NEUMANN (II.), as also by HAMMARSTEN (III.), ALFR. NOLL (I.), and by I. BANG (I.). The two first-named workers, however, have also shown that many nucleins contain two kinds of carbohydrates: non-reducing carbohydrates present in the molecule of the nucleic acid; and reducing carbohydrates which, along with the latter substance, take part in the structure of the nuclein complex, and appear in company with nucleic acid on the decomposition of the main substance.

Of the nuclein bases here in question, the first to be discovered in the manner described above was hypoxanthin (CHANO), which was obtained from yeast nuclein by KOSSEL (III.) in 1879. Two years later he prepared 10 grams of this base direct from pressed yeast by a method (VII.) of his own,

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which he afterwards improved (IX.), and showed (VIII.) that the same is also obtainable from nucleins of different (animal) origin. In the meantime he isolated (III.) from yeast nuclein a second base, xanthin (CHNO2), to which he added a third, namely guanin (CHN ̧O) in 1882 (IX.). The occurrence of the lastnamed in yeast has also been placed beyond doubt by S. SCHINDLER (I.). These three allies of uric acid (C,H,N,O,) had already been long known to chemists, though not as constituents of nucleins. On the other hand, the four additional bases mentioned below were all new, and were obtained for the first time from the nucleins. Adenin (C,H,N) was isolated by KOSSEL (XI.) in 1885, first from the nuclein of ox pancreas and afterwards from pressed yeast. This substance stands in the same relation towards hypoxanthin as guanin does to xanthin, not only as far as chemical composition is concerned, but also in respect of behaviour during putrefaction caused by certain bacteria, which, according to the researches of S. SCHINDLER (I.), degrade guanin to xanthin, and adenin to hypoxanthin. According to A. BAGINSKY (IV.), the last-named offers the greater resistance to the influences in question. In 1893 another new base, namely thymin, was brought to our knowledge by A. KOSSEL and A. NEUMANN (I.). It was first obtained from the nucleic acid isolated from the thymoid gland of the calf, which acid received the name of adenylic acid on account of its capacity for furnishing adenin. The thymin molecule (which has the formula C.H.NO) may be regarded as methyl-dioxy-pyrimidin, in conformity with the views of H. STEUDEL (I.), or as 5-methyl-uracyl, in sympathy with those of E. FISCHER and G. REDER (I.), who were the first to prepare it by synthetic means. The hypothesis raised by KOSSEL and NEUMANN (II.) that thymin is also present in the molecular complex of the nucleic acid of yeast, has not been confirmed, though, according to A. ASCOLI (III.), the parent substance of thymin, namely uracyl (CHN2O2) is found therein. It may also be remarked by the way that the fourth of the new nuclein bases, cytosin, has so far only been found as a constituent of nucleic acids of animal origin. In the crystalline state this substance has the formula C2H30N1604 5H2O.

Apart from the last-named, which has not yet been sufficiently investigated, the nuclein bases mentioned may be classified into two groups in accordance with their constitution. Adenin, xanthin, hypoxanthin, and guanin are derived from the atomic complex to which EMIL FISCHER (I.) gave the name Purin :