This result is probably to be explained, at all events in the main, by the presence in the co-enzyme solution of the antiprotease to which reference has already been made [Buchner and Haehn, [1910, 2]]. This agent, the constitution of which is still unknown, protects proteins in general from the action of digestive enzymes, and on the assumption that the alcoholic enzyme of yeast-juice belongs to the class of proteins, may be supposed to lessen the rate at which this enzyme is destroyed by the endotryptase of the juice. This antiprotease is, like the co-enzyme (p. [68]), destroyed by lipase but is more stable than the co-enzyme towards hydrolytic agents, and can be obtained free from co-enzyme by boiling the solution for some hours alone or by heating with dilute sulphuric acid. Such a solution possesses no regenerative power, but still retains its power of protecting proteins against digestion and of preserving the fermenting power of yeast-juice. [p066]
It must, however, be remembered that the addition of a phosphate alone may greatly prolong the period of fermentation of yeast-juice (p. [55]), and sugar is well known to exert a similar action. It appears, therefore, that the existence of the enzyme is prolonged not only by the presence of the antiprotease but also by that of sugar and hexosephosphate, into which phosphate passes in presence of sugar. Similar effects are exerted on the co-enzyme by sugar and probably also by hexosephosphate.
The fermenting complex, therefore, in the presence of these substances, either separately or together, falls off more slowly in activity and is present for a longer time, and for both of these reasons produces an increased amount of fermentation. It seems probable also that the hexosephosphatase is similarly affected, so that the supply of free phosphate is at the same time better maintained, and the rate of fermentation for this reason decreases more slowly than would otherwise be the case.
It is in this way that an explanation may be found of the remarkable increase in total fermentation, which is produced by the addition to yeast-juice and sugar of boiled yeast-juice, containing free phosphate (which passes into hexosephosphate) as well as co-enzyme, of boiled autolysed yeast-juice, containing free phosphate but no co-enzyme, or of phosphate solution alone.
In no case is the original rate of fermentation greatly increased after the initial acceleration has disappeared, but in every case the total fermentation is considerably augmented, and this is no doubt mainly to be attributed, as just explained, to the diminished rate of decomposition of the fermenting complex and probably of the hexosephosphatase.
Although both enzyme and co-enzyme are completely precipitated from yeast-juice, as already described (p. [38]), by 10 volumes of acetone, the co-enzyme is less easily precipitated than the enzyme, and a certain degree of separation can therefore be attained by fractional precipitation [Buchner and Duchaček, [1909]]. The enzyme cannot, however, be completely freed from co-enzyme in this manner, and the process is attended by a very considerable loss of enzyme. This is probably due to the fact that only small quantities of acetone can be added (1·5 to 3 volumes), in order to avoid precipitation of co-enzyme, and that the precipitates thus formed contain a large proportion of water, a condition which appears to be fatal to the preservation of the enzyme.
It is, however, not quite certain whether it is the zymase or the hexosephosphatase which is destroyed in these cases, as no attempt [p067] was made to distinguish between them. In any case the precipitates obtained by fractional treatment with acetone, even when reunited, produce a much smaller fermentation than the original juice or the powder prepared by bringing it into 10 volumes of acetone.
Attempts to isolate the co-enzyme from boiled yeast-juice have also been hitherto unsuccessful. It has, however, been found possible to remove a considerable amount of material from the solution without affecting the co-enzyme. When 1 volume of alcohol is added to boiled yeast-juice, a bulky precipitate, consisting largely of carbohydrates, is produced, and the filtrate from this is found to contain the co-enzyme and can be freed from alcohol by evaporation. Further precipitation with alcohol has not led to useful results.
When a solution which has been treated in this way is precipitated with lead acetate and kept neutral to litmus, the free phosphate and hexosephosphate are thrown down and the co-enzyme remains in solution. The filtrate can be freed from lead by means of sulphuretted hydrogen and neutralised, and then forms a solution of co-enzyme free from phosphate and hexosephosphate but still containing combined phosphorus. More complete purification than this has not yet been accomplished. Occasionally the precipitate of lead salts retains some of the co-enzyme, apparently by adsorption, but usually the greater part remains in the solution (Harden and Young).
The co-enzyme is partially removed from yeast-juice by means of a colloidal solution of ferric hydroxide (Resenscheck). A precipitate is thus obtained which contains phosphorus and resembles boiled yeast-juice in its regenerative action on yeast-juice rendered inactive by fermentation. It has not, however, so far been found possible to isolate any definite compound from this precipitate. There are also indications that when yeast-juice, either fresh or boiled, is electrolysed, the co-enzyme tends to accumulate at the cathode [Resenscheck, [1908, 1], [2]].