I. Influence of Concentration of Phosphate on the Course of Fermentation.
Prominent among these instances of inhibition and acceleration are the phenomena attendant on the addition of excess of phosphate to yeast-juice.
When a phosphate is added to a fermenting mixture of a sugar and yeast-juice, the effect varies with the concentration of the phosphate and the sugar and with the particular specimen of yeast-juice employed. With low concentrations of phosphate in presence of excess of glucose the acceleration produced is so transient that no accurate measurements of rate can be made. As soon as the amount of phosphate added is sufficiently large, it is found that the rate of evolution of carbon dioxide very rapidly increases from five to ten times, and then quickly falls approximately to its original value.
As the concentration of phosphate is still further increased, it is first observed that the maximum velocity, which is still attained almost immediately after the addition of the phosphate, is maintained for a certain period before the fall commences, and then, as the increase in concentration of phosphate proceeds, that the maximum is only gradually attained after the addition, the period required for this increasing with the concentration of the phosphate. Moreover, with still higher concentrations, the maximum rate attained is less than that reached with lower concentrations, and further, the rate falls off more slowly. The concentration of phosphate which produces the highest rate, which may be termed the optimum concentration, varies very considerably with different specimens of yeast-juice [Harden and Young, [1908, 1]].
All these points are illustrated by the accompanying curves (Fig. 7) which show the rate of evolution per five minutes plotted against the time for four solutions in which the initial concentrations of phosphate were (A) 0·033, (B) 0·067, (C) 0·1, and (D) 0·133 molar, the volumes of 0·3 molar phosphate being 5, 10, 15, and 20 c.c. in each case added to 25 c.c. of yeast-juice, and made up to 45 c.c, each solution containing 4·5 grams of glucose. The time of addition is taken as zero, the rate before addition being constant, as shown in the curves.
Fig. 7.
It will be observed that 5 and 10 c.c. (A and B) give the same maximum, whilst 15 c.c. (C) produce a much lower maximum, and 20 c.c. (D) a still lower one, the rate at which the velocity diminishes after the attainment of the maximum being correspondingly slow in these last two cases. By calculating the amount of phosphate which has disappeared as such from the amount of carbon dioxide evolved, [p072] it is found that the maximum does not occur at the same concentration of free phosphate in each case.
These results suggest that the phosphate is capable of forming two or more different unstable associations with the fermenting complex. One of these, formed with low concentrations of the phosphate, has the composition most favourable for the decomposition of sugar, whilst the others, formed with higher concentrations of phosphate, contain more of the latter, probably associated in such a way with the fermenting complex as to render the latter partially or wholly incapable of effecting the decomposition of the sugar molecule. As the fermentation proceeds slowly in the presence of excess of phosphate, the concentration of the latter is reduced by conversion into hexosephosphate, and a re-distribution of phosphate occurs, resulting in the gradual change of the less active into the more active association of phosphate with fermenting complex, and a consequent rise in the rate of fermentation.
In those cases in which the maximum rate corresponding to the optimum concentration of phosphate is never attained, some secondary cause may be supposed to intervene, such as a permanent change in a portion of the fermenting complex, accumulation of the products of the reaction, etc.
It is also possible as suggested by Buchner for the analogous case of arsenite (p. [78]) that the addition of increasing amounts of phosphate causes a progressive but reversible change in the mode of dispersion [p073] of the colloidal enzyme, and that this has the secondary effect of altering the rate of fermentation. No decisive evidence is as yet available upon the subject.
The results obtained by Euler and Johansson [[1913]] to which reference has already been made indicate that in presence of a moderate excess of phosphate esterification is more rapid than production of carbon dioxide. No explanation of this phenomenon has yet been given, but it might obviously be due either to the production of some phosphorus compound which subsequently takes part in the production both of hexosediphosphate and of carbon dioxide, or, less probably, to the entire independence of the two changes—esterification of phosphate and production of carbon dioxide—which might then be differently affected by the presence of excess of phosphate and therefore take place at different rates.