CHAPTER III.

THE FUNCTION OF PHOSPHATES IN ALCOHOLIC FERMENTATION.

In the course of some preliminary experiments (commenced by the late Allan Macfadyen, but subsequently abandoned) on the production of anti-ferments by the injection of yeast-juice into animals, the serum of the treated animals was tested for the presence of such antibodies both for the alcoholic and proteoclastic enzymes of yeast-juice, and it was then observed that the serum of normal and of treated animals alike greatly diminished the autolysis of yeast-juice.

As the explanation of the comparatively rapid disappearance of the fermenting power from yeast-juice had been sought, as already mentioned (p. 20), in the hydrolytic action of the tryptic enzyme which always accompanies zymase, the experiment was made of carrying out the fermentation in the presence of serum, with the result that about 60 to 80 per cent. more sugar was fermented than in the absence of the serum [Harden, 1903].

This fact was the starting-point of a series of attempts to obtain a similar effect by different means, in the course of which a boiled and filtered solution of autolysed yeast-juice was used, in the hope that the products formed by the action of the tryptic enzyme on the proteins of the juice would, in accordance with the general rule, prove to be an effective inhibitant of that enzyme. This solution was, in fact, found to produce a very marked increase in the total fermentation effected by yeast-juice, the addition of a volume of boiled juice equal to that of the yeast-juice doubling the amount of carbon dioxide evolved [Harden and Young, 1905, 1]. This effect was found to be common to the filtrates from boiled fresh yeast-juice and from boiled autolysed yeast-juice, and was ultimately traced in the main, not to the antitryptic effect which had been surmised, but to two independent factors, either of which was capable in some degree of bringing about the observed result.

Boiled yeast-juice was indeed found to possess a decided anti-autolytic effect, as determined by a comparison of the amounts of nitrogen rendered non-precipitable by tannic acid in yeast-juice alone [p042] and in a mixture of yeast-juice and boiled juice on preservation [Harden, 1905]. The anti-autolytic effect, however, appeared to vary independently of the effect on the fermentation, and the conclusion was drawn, as stated above, that the increase in the alcoholic fermentation was not directly dependent on the decrease in the action of the proteoclastic enzyme but was due to some independent cause. The property possessed by boiled yeast-juice of diminishing the autolysis of yeast-juice has now been carefully examined by Buchner and Haehn [1910, 2] and ascribed by them to a soluble antiprotease (p. 65).

The two factors to which the increase in fermentation produced by the addition of boiled juice were ultimately traced were (1) the presence of phosphates in the liquid, and (2) the existence in boiled fresh yeast-juice of a co-ferment or co-enzyme, the presence of which is indispensable for fermentation [Harden and Young, 1905, 1, 2].

The former of these factors will be here discussed and the co-enzyme will form the subject of the following chapter.

The general fact that sodium phosphate increases the total fermentation produced by a given volume of yeast juice was observed on several occasions by Wroblewski [1901] and also by Buchner [Buchner, E. and H., and Hahn, 1903, pp. 141-2], who ascribed the action of this salt to its alkalinity, comparing it in this respect with potassium carbonate and remarking that the increase in both cases took place chiefly in the first twenty hours of fermentation. The increased amount of fermentation following the addition of boiled yeast-juice was also noted by Buchner and Rapp [1899, 2, No. 265, p. 2093] in a single experiment.

Observations made at intervals of a few minutes instead of twenty hours have, however, revealed the fact that phosphates play a part of fundamental importance in alcoholic fermentation and that their presence is absolutely essential for the production of the phenomenon.

EFFECT OF THE ADDITION OF PHOSPHATE TO A FERMENTING MIXTURE OF YEAST-JUICE AND SUGAR.

When a suitable quantity[2] of a soluble phosphate is added to a fermenting mixture of glucose, fructose, or mannose with yeast-juice, the rate of fermentation rapidly rises, sometimes increasing as much as twenty-fold, continues at this high value for a certain period and then falls again to a value approximately equal to, but generally [p043] somewhat higher than, that which it originally had. Careful experiments have shown that during this period of enhanced fermentation the amounts of carbon dioxide and alcohol produced exceed those which would have been formed in the absence of added phosphate by a quantity exactly equivalent to the phosphate added in the ratio CO{2} or C{2}H{6}O:R′{2}HPO{4} [Harden and Young, 1906, 1].

[2] The effect of an excess of phosphate is discussed later on, p. 71.

This result is of fundamental importance, and the evidence on which it rests deserves some consideration. Quantitative experiments on this subject require certain preliminary precautions. The acid phosphates are too acid to permit of any extended fermentation and the phosphates of the formula R′{2}HPO{4} absorb a considerable volume of carbon dioxide with production of a bicarbonate, according to the reaction:--

R{2}HPO{4} + H{2}CO{3} ⇌ RHCO{3} + RH{2}PO{4}.

The method which has been adopted, therefore, is to employ either a secondary phosphate saturated with carbon dioxide at the temperature of the experiment, or a mixture of five molecular proportions of the secondary phosphate with one molecular proportion of a primary phosphate, in which the amount of bicarbonate formed is negligible. In the former case it is necessary to ascertain whether any of the carbon dioxide evolved is derived from the bicarbonate by the action of acid originally present or produced in the yeast-juice or by a disturbance of the original equilibrium owing to the chemical change which occurs. This is done by acidifying duplicate samples with hydrochloric acid before and after the fermentation and measuring the gas evolved in each case. Any necessary correction can then be made. The calculation of the extra amount of carbon dioxide evolved from yeast-juice containing sugar when a phosphate is added involves an estimation of the amount which would have been evolved in the absence of added phosphate, and this is a matter of some difficulty. Since the final steady rate of fermentation attained is often slightly different from the initial rate, the practice has been adopted of ascertaining this final rate and then calculating the total evolution corresponding to it for the whole period from the time of the addition of the phosphate to the end of the observations. This amount deducted from the observed total leaves the extra amount of carbon dioxide formed, and it is this quantity which is equivalent to the phosphate added. Alcohol is simultaneously produced in the normal ratio. The justification for this method of calculation will be found later (p. 54).

The following table, containing the results of experiments with [Pg 044] glucose, fructose, and mannose, indicates very clearly the nature of the method of calculation and also of the agreement between observation and theory.

Three quantities of 25 c.c. of yeast-juice + 5 c.c. of a solution containing 1 gram of the sugar to be examined (a large excess) were incubated with toluene at 25° for one hour, in order to remove all free phosphate, and to each were then added 5 c.c. of a solution of sodium phosphate corresponding to 0·1632 gram of Mg{2}P{2}O{7} and equivalent to 32·6 c.c. of carbon dioxide at N.T.P. The rates of fermentation were then observed until they had passed through the period of acceleration and had fallen and attained a steady value, the gases being measured moist at 19·3° and 760·15 mm.

─────────────────────────────────────────────────┬───────────────────── │ Glucose. │ ┌────────────── │ │ Mannose. │ │ ┌───────── │ │ │Fructose. ─────────────────────────────────────────────────┼──────┼────┴───────── Maximum rate attained, c.cs. per five minutes │ 9·6 │ 7 11·3 Final rate of fermentation │ 1·1 │ 0·96 1·08 Total carbon dioxide produced by fermentation in │ │ fifty-five minutes after addition of phosphate│ 49·7 │ 47·8 47·6 Correction for evolution in absence of phosphate │ │ in fifty-five minutes │ 12·1 │ 10·6 11·9 Extra carbon dioxide equivalent to phosphate │ 37·6 │ 37·2 35·7 " " " " " " at N.T.P.│ 34·4 │ 34 32·6 ─────────────────────────────────────────────────┴──────┴──────────────

These numbers agree well with the value calculated from the phosphate added, viz. 32·6 [Harden and Young, 1909].

Another experiment is illustrated graphically in Fig. 4, in which the volume of carbon dioxide evolved is plotted against time. The determination was in this case made by adding 25 c.c. of an aqueous solution containing 5 grams of glucose to one quantity of 25 c.c. of yeast-juice (curve A) and 5 c.c. of 0·3 molar solution of the mixed primary and secondary sodium phosphates, and 20 c.c. of a solution containing 5 grams of glucose to a second equal quantity of yeast-juice (curve B). Curve A shows the normal course of fermentation of yeast-juice with glucose. There is a slight preliminary acceleration during the first twenty minutes, due to free phosphate in the juice, and the rate then becomes steady at about 1·4 c.c. in five minutes. During this preliminary acceleration 10 c.c. of extra carbon dioxide are evolved, this number being obtained graphically by continuing the line of steady rate back to the axis of zero time. Curve B shows the effect of the added phosphate. The rate rises to about 9·5 c.c. in five minutes, i.e. to more than six times the normal rate, and then gradually falls until after an hour it is again steady and almost exactly equal to 1·4 c.c. per five minutes. Continuing the line of steady rate back to the axis of zero [p045] time it is found that the extra amount of carbon dioxide is 48 c.c. Subtracting from this the 10 c.c. shown in curve A as due to the juice alone, a difference of 38 c.c. is obtained due to the added phosphate. The amount calculated from the phosphate added in this case is, at atmospheric temperature and pressure, 38·9 c.c.

After the expiration of seventy minutes from the commencement of the experiment, a second addition is made of an equal amount of phosphate. The whole phenomenon then recurs, as shown in curve C, the maximum rate being slightly lower than before, about 6 c.c. per five minutes, and the rate again becoming finally steady at 1·4 c.c. as before. The extra amount of carbon dioxide evolved in this second period obtained graphically as in the former case, is 107-68 = 39 c.c.

It may be noted that in this case the observations after each addition last fifty to seventy minutes, so that an error of 0·1 c.c. per five minutes in the estimated final rate would make an error of 1 to 1·4 c.c. in the extra amount of carbon dioxide, i.e. about 3 to 4 per cent. of the total, and this is approximately the limit of accuracy of the method. [p046] The results are more precise when the yeast-juice employed is an active one, since, when the fermenting power of the juice is low, the initial period of accelerated fermentation is unduly prolonged and the calculation of the extra amount of carbon dioxide is rendered uncertain.

Zymin (p. 38) yields precisely similar results to yeast-juice, but in this case the rate of fermentation is not so largely increased. This has the effect that the extra amount of carbon dioxide cannot be quite so accurately estimated for zymin, because a slight error in the determination of the final rate of fermentation has a greater influence on the result. The equivalence between the extra amount of carbon dioxide evolved and the phosphate added is, however, unmistakable, as is shown by the following results of an experiment with zymin, in which 6 grams of zymin (Schroder) + 3 grams of fructose (Schering) + 25 c.c. of water were incubated at 25° in presence of toluene until a steady rate had been attained. Five c.c. of a solution of sodium phosphate equivalent to 32·2 c.c. carbon dioxide at N.T.P. were then added.

Maximum rate attained, c.c. per five minutes 14·1

Final rate of fermentation 6·2

Total evolved by fermentation in eighty minutes after addition of phosphate 131

Correction for evolution in absence of phosphate in eighty minutes 99·2

Extra carbon dioxide at 16° and 767·1 mm 31·8

" " " " N.T.P 29·8

Considering the small proportional rise in rate and the long period of accelerated fermentation, the agreement between the volume observed, 29·8 c.c., and that calculated from the phosphate, 32·2, is quite satisfactory [Harden and Young, 1910, 1.] Precisely the same relations hold for maceration extract, but in this case it must be remembered that a large amount of free phosphate is present in the extract, as much as 0·3129 grm. Mg{2}P{2}O{7} being obtained from 20 c.c. in one preparation, so that the original extract had the concentration of a 0·14 molar solution of sodium phosphate. It is in fact not improbable that the delay in the onset of fermentation sometimes observed with maceration extract (see Lebedeff, 1912, 2; Neuberg and Rosenthal, 1913) may be due to the presence of phosphate in so great an excess of the amount which can be rapidly esterified by the enzymes that the rate of fermentation is at first greatly lowered (see p. 71). When this phosphate is removed by incubation with glucose or fructose, the subsequent addition of phosphate produces the characteristic action and the extra carbon dioxide evolved is, as with other yeast preparations, equivalent to the phosphate added. An actual estimation carried out in this way gave 35 c.c. of CO{2} for an addition of phosphate equivalent to 32·9 c.c. [Harden and Young, 1912]. [p047]

Within the limits imposed by the experimental conditions, then, the fact is well established that the addition of a soluble phosphate to a fermenting mixture of a hexose with yeast-juice, maceration extract, dried yeast, or zymin causes the production of an equivalent amount of carbon dioxide and alcohol.

This fact indicates that a definite chemical reaction occurs in which sugar and phosphate are concerned, and this conclusion is confirmed when the fate of the added phosphate is investigated. If an experiment, such as one of those described above, be interrupted as soon as the rate of fermentation has again become normal, and the liquid be boiled and filtered, it is found that nearly the whole of the phosphorus present passes into the filtrate, but that only a small proportion of this exists as mineral phosphate, whilst the remainder, including that added in the form of a soluble phosphate, is no longer precipitable by magnesium citrate mixture [Harden and Young, 1905, 2].

A similar observation was made at a later date by Iwanoff [1907], who had previously observed [1905] that living yeast, like many other vegetable organisms, converted mineral phosphates into organic derivatives. Iwanoff employed zymin and hefanol (p. 38) instead of yeast-juice, and found that phosphates were thereby rendered non-precipitable by uranium acetate solution, but did not observe the accelerated fermentation caused by their addition.

The foregoing conclusions have been strikingly confirmed by experiments with maceration extract carried out by Euler and Johansson [1913], in which both the carbon dioxide evolved and the phosphate rendered non-precipitable by magnesia were determined at intervals. When dried yeast is employed as the fermenting agent, the amount of phosphate esterified in the earlier stages is greater than would be expected, but ultimately becomes exactly equivalent to the carbon dioxide evolved.

NATURE OF THE PHOSPHO-ORGANIC COMPOUND FORMED BY YEAST-JUICE AND ZYMIN FROM THE HEXOSES AND PHOSPHATE.

The formation and properties of the compound produced from phosphates in the manner just described have been investigated by Harden and Young [1905, 2; 1908, 1; 1909; 1911, 2], Young [1909; 1911], Iwanoff [1907; 1909, 1], Lebedeff [1909; 1910; 1911, 5, 6; 1912, 3; 1913, 1]; and Euler [1912, 1; Euler and Fodor, 1911; Euler and Kullberg, 1911, 3; Euler and Ohlsén, 1911; 1912; Euler and Johansson, 1912, 4; Euler and Bäckström, 1912], but its exact constitution cannot as yet be regarded as definitely known. [p048]

Phosphates undergo this characteristic change when the sugar undergoing fermentation is glucose, mannose, or fructose, and it may be said at once that no distinction can be established between the products formed from these various hexoses; they all appear to be identical. The compound produced is, as already mentioned, not precipitated by ammoniacal magnesium citrate mixture, nor by uranium acetate solution. It can, however, be precipitated by copper acetate (Iwanoff) and by lead acetate (Young). The preparation of the pure lead salt from the liquid obtained by fermenting a sugar with yeast-juice or zymin in presence of phosphate is commenced by boiling and filtering the liquid. Magnesium nitrate solution and a small quantity of caustic soda solution are then added to precipitate any free phosphate, and the liquid well stirred and allowed to stand over night. To the neutralised filtrate lead acetate is then added together with sufficient caustic soda solution to maintain the reaction neutral to litmus, until no further precipitate is formed. The liquid is then filtered or, better, centrifugalised, and the precipitate repeatedly washed with water until a portion of the clear filtrate gives no reduction when boiled with Fehling's solution. It is essential that this washing should be thorough as evidence has recently been obtained of the formation under certain conditions of a hexosephosphate, the lead salt of which is not so sparingly soluble as that of the hexosediphosphate [Harden and Robison, 1914]. The lead precipitate is then suspended in water, decomposed by a current of sulphuretted hydrogen, the clear filtrate freed from sulphuretted hydrogen by a current of air, and finally neutralised with caustic soda. The removal of phosphate and conversion into lead salt are repeated twice, and the resulting lead salt is then found to be free from nitrogen and to have a composition represented by the formula C{6}H{10}O{4}(PO{4}Pb){2}. Lebedeff carries out the preparation in a somewhat different manner. The fermentation is effected by means of air-dried yeast (150 grams to 1 litre of water, 210 grams cane-sugar and 105 grams of a mixture of 2 parts Na{2}HPO{4} and 1 part NaH{2}PO{4}) and the liquid (about 700 c.c.) after boiling and filtering, is treated with an equal volume of acetone. About 300 c.c. of a thick liquid is precipitated and this is redissolved in water and precipitated by an equal volume of acetone two or three times. The final liquid is then precipitated with warm lead acetate solution and filtered and washed with dilute lead acetate solution until the filtrate is clear and no longer reduces Fehling's solution after removal of the lead [1910]. Euler and Fodor [1911] on the other hand precipitate the free phosphate with magnesia mixture and then add acetone, dissolve the syrup thus precipitated in water and add copper [p049] acetate solution. A blue copper salt is precipitated which is thoroughly washed with water and used for the preparation of solutions of the acid. A solution of the free acid can readily be prepared by the action of sulphuretted hydrogen on the lead salt suspended in water. It forms a strongly acid liquid, which requires exactly two equivalents of base for each atom of phosphorus present to render it neutral to phenolphthalein. It decomposes when evaporated, leaving a charred mass containing free phosphoric acid. The acid is slightly optically active, and has [/a/{D}] = + 3·4°. A number of amorphous salts have been prepared by precipitation from a solution of the sodium salt, and of these the silver, barium, and calcium salts have been analysed with results agreeing with the general formula C{6}H{10}O{4}(PO{4}R′{2}){2}. The magnesium, calcium, barium, and manganese salts, which are only sparingly soluble, are all precipitated when their solutions are boiled but re-dissolve on cooling, and this property can be utilised for their purification. The alkali salts have only been obtained as viscid residues.

A difference of opinion exists as to the molecular weight and constitution of this substance. Iwanoff [1909, 1] regards it as a triosephosphoric acid, C{3}H{5}O{2}(PO{4}H{2}), basing this view on the preparation of an osazone which melted at 142°, but when recrystallised from benzene gave a product melting at 127°-8°, which had the same appearance, melting-point, and nitrogen content as the triosazone formed by the action of phenylhydrazine on the oxidation products of glycerol. Neither Lebedeff [1909] nor Young could obtain Iwanoff's osazone, and all attempts to reduce the acid with formation of glycerol either by sodium amalgam or hydriodic acid were unsuccessful (Young). There is therefore practically no serious experimental evidence in favour of Iwanoff's view.

On the other hand, Harden and Young regard the acid as a diphosphoric ester of a hexose. This view is based on the fact that when the acid is boiled with water, or an acid, free phosphoric acid is produced along with a levo-rotatory solution containing fructose and possibly a small proportion of some other sugar or sugars. (Euler and Fodor however did not obtain a hexose in this way [1911].) The acid itself only reduces Fehling's solution after some hours in the cold, rapidly when boiled, whereas when its solution is first boiled, and then treated with Fehling's solution in the cold, the products of decomposition bring about reduction in a few minutes. The reduction brought about when the acid is boiled with Fehling's solution is considerably less (33 per cent.) than that produced by an equivalent amount of glucose. The behaviour of the compound towards phenylhydrazine is also in complete agreement [p050] with this view. Lebedeff found [1909, 1910] that the acid or its salts heated with phenylhydrazine in presence of acetic acid gave an insoluble compound which was ultimately found to be the /phenylhydrazine salt of hexosemonophosphoric acid osazone/

C{6}H{5}NH·NH{2}·H{2}PO{4}·C{4}H{5}(OH){3}· C(N{2}HC{6}H{5})CH(N{2}HC{6}H{5})

[Lebedeff, 1910; 1911, 6; Young, 1911]. After recrystallisation from alcohol this compound forms yellow needles, melting at 151°-152°. It is decomposed by caustic soda yielding a /sodium salt/

Na{2}PO{4}·C{4}H{5}(OH){3}·(CN{2}HC{6}H{5})·CH(N{2}HC{6}H{5})

and on boiling with caustic soda decomposes giving a hexosazone (free from phosphorus) which is probably glucosazone, and in addition glyoxalosazone, probably as the result of a secondary decomposition. Towards acids it is remarkably stable yielding with hydrochloric acid a /hexosonephosphoric ester/ from which the original osazone can be regenerated (Lebedeff). Lebedeff at first [1910] argued from the formation of this osazone that the original hexosephosphate contained only one phosphoric acid group per molecule of hexose. It was however shown by Young [1911] and subsequently confirmed by Lebedeff [1911, 6] that one molecule of phosphoric acid is split off during the formation of the osazone, even in neutral solution. Moreover it has been found that in the cold hexosediphosphoric acid reacts with 3 molecules of phenylhydrazine forming the /diphenylhydrazine salt of hexosediphosphoric acid phenylhydrazone/

(C{6}H{5}NH·NH{2}·H{2}PO{4}){2}·C{6}H{7}(OH){3}·N{2}HC{6}H{5}.

This compound crystallises out when 1 volume of alcohol is added to a solution of 3 molecules of phenylhydrazine in one of the acid and forms colourless needles melting at 115°-117°. p-Bromophenylhydrazine yields an analogous compound melting at 127°-128°.

Precisely the same products are given with phenylhydrazine by the hexosephosphoric acid prepared from glucose, mannose, and fructose, proving that all these sugars yield the same hexosediphosphoric acid, a point of fundamental importance.

Direct measurements of the molecular weight of the acid by the freezing-point method, combined with the determination of the degree of dissociation by the rate of cane-sugar inversion, are indecisive, but indicate that the acid has a molecular weight considerably higher than that required for a triosephosphoric acid.

A similar uncertainty attaches to the determination of the molecular weight from the freezing-point depression and conductivity of the acid potassium salt [Euler and Fodor, 1911]. Euler however concludes [p051] that both a hexosediphosphoric acid and a triosemonophosphoric acid are formed, but has not prepared any derivatives of the latter.

As regards the constitution of the hexosephosphoric ester several suggestions have been made by Young, but no decisive evidence at present exists. The identity of the products from glucose, mannose, and fructose may be explained by regarding the acid as a derivative of the enolic form common to these three sugars (p. 97), or by supposing that portions of two sugar molecules may be concerned in its production. The formation and composition of the hydrazone and osazone are of great importance as they indicate that in all probability one of the phosphoric acid residues is united with the carbon atom adjacent to the carbonyl group of the hexose. They moreover render it certain that the original phosphoric ester is a hexosediphosphoric ester and not a triosemonophosphoric ester.

Hexosediphosphoric acid has not as yet been discovered in the animal body. The action of a number of enzymes upon it has been examined [Euler, 1912, 2; Euler and Funke, 1912; Harding, 1912; Plimmer, 1913] with the following results.

The lipase of castor oil seeds, a glycerol extract of the intestinal mucous membrane of the rabbit and pig, and an aqueous extract of bran have a slow hydrolytic action, whereas pepsin and trypsin are without effect. Feeding experiments with rabbits and dogs indicate that the ester is capable of hydrolysis in the animal body, a large proportion of the phosphorus being excreted as inorganic phosphate. The ester is also decomposed by /Bacillus coli communis/.

It is remarkable that the hexosephosphate is not fermented nor hydrolysed by living yeast, a fact observed by Iwanoff, Harden and Young, and Euler, although, according to the experiments of Paine [1911], the yeast cell is at all events partially permeable to the sodium salt.

THE EQUATION OF ALCOHOLIC FERMENTATION.

An equation can readily be constructed for the reaction in which hexosephosphate is formed, the data available being the formula of the product and the relation between the phosphate added and the carbon dioxide and alcohol produced:--

(1) 2C{6}H{12}O{6} + 2PO{4}HR{3} = 2CO{2} + 2C{2}H{6}O + 2H{2}O + C{6}H{10}O{4}(PO{4}R{2}){2}.

According to this, two molecules of sugar are concerned in the change, the carbon dioxide and alcohol being equal in weight to one [p052] half of the sugar used, and the hexosephosphate and water representing the other half.

Additional confirmation of this equation is afforded by the determination of the ratio between sugar used and carbon dioxide evolved when a known weight of sugar together with an excess of phosphate is added to yeast-juice at 25°. The phenomena then observed are precisely similar to those which occur when a phosphate is added to a fermenting mixture of yeast-juice and excess of sugar as described above. The rate of fermentation rapidly rises and then gradually falls until a rate is attained approximately equal to that of the autofermentation of the juice in presence of phosphate. At this point it is found that the extra amount of carbon dioxide evolved, beyond that which would have been given off in the absence of added sugar, bears the ratio expressed in equation (1) to the sugar added [Harden and Young, 1910, 2]. The results of four estimations made in this way were (/a/) 0·2 grams of glucose gave 26·5 and 27·9 c.c. of carbon dioxide at N.T.P.; (/b/) 0·2 grams of fructose gave 27·9 and 28·9 c.c. The carbon dioxide calculated from the sugar added in each of the four cases is 26·96 c.c.

It has also been shown by Euler and Johansson [1913] that in the fermentation of a mixture of equivalent amounts of phosphate and glucose, the whole of the glucose had disappeared when the whole of the phosphate had become esterified.

CYCLE OF CHANGES UNDERGONE BY PHOSPHATE IN ALCOHOLIC FERMENTATION.

According to equation (1) the free phosphate present is used up in the reaction, and the question at once arises whether there is any source from which a constant supply of free phosphate can be elaborated in the juice, or whether some other change occurs which results in the formation of carbon dioxide and alcohol in the absence of free phosphate. The experimental evidence points in the direction of the former of these alternatives, but the question is a very difficult one to decide with absolute certainty.

When a mixture of a phosphate with yeast-juice and sugar is examined at intervals and the amount of free phosphate estimated, the following stages are observed:--

1. During the initial period of accelerated fermentation following the addition of the phosphate, the concentration of free phosphate rapidly diminishes.

2. At the close of this period, the amount of free phosphate [p053] present is very low, and, as long as active fermentation continues, no marked increase in it occurs.

3. As alcoholic fermentation slackens and finally ceases, a marked and rapid rise in the amount of free phosphate occurs at the expense of the hexosephosphate, which steadily diminishes in amount, and this change is brought about by an enzyme in the juice and ceases if the liquid be boiled.

This last reaction may be represented by the equation

(2) C{6}H{10}O{4}(PO{4}R{2}){2} + 2H{2}O = C{6}H{12}O{6} + 2PO{4}HR{2}.

In the light of this equation, together with equation No. 1, given above, all these facts can be simply and easily understood.

The rapid diminution in the amount of free phosphate during stage 1 corresponds with the occurrence of reaction (1). During the whole period of fermentation the enzymic hydrolysis of the hexosephosphate is proceeding according to equation (2). Up to the end of stage 2 the phosphate thus produced enters into reaction, according to equation (1), with the sugar which is present in excess and is thus reconverted into hexosephosphate, so that as long as alcoholic fermentation is proceeding freely, no accumulation of free phosphate can occur.

As soon as alcoholic fermentation ceases, however, it is no longer possible for the phosphate to pass back into hexosephosphate, and hence it accumulates in the free state.

A similar hydrolysis of hexosephosphate and accumulation of phosphate occur when a solution of hexosephosphate is treated with yeast-juice which has been deprived of the power of fermentation by dialysis, or with zymin freed from co-enzyme by washing (p. 63).

The actual rate of fermentation observed in any particular case in presence of excess of sugar, enzyme, and co-enzyme must on this view depend on the supply of phosphate which is available.

In presence of an adequate amount of phosphate, as well as of sugar, the highest rate attained represents the maximum velocity at which reaction (1) can proceed in that sample of yeast-juice or zymin, and this high rate is characteristic of the initial period of accelerated fermentation which follows the addition of a suitable quantity of phosphate. By the simple expedient of renewing the supply of phosphate as rapidly as it is converted into hexosephosphate, this high rate can be maintained for a considerable time [Harden and Young, 1908, 1]. In this way, for example, an average rate of evolution of carbon dioxide of 15 c.c. in five minutes was maintained for an hour and a [p054] quarter, whereas the normal rate in the absence of added phosphate was 3 c.c.

As soon as all the free phosphate has entered into the reaction, however, the supply of phosphate depends in the main on the rate at which the resulting hexosephosphate is decomposed, and the rate of fermentation now attained is conditioned by the rate at which reaction (2) proceeds, and this evidently depends on the existing concentration of the hydrolytic enzyme, which may be provisionally termed /hexosephosphatase/.

The rates attained during the initial period of rapid fermentation and the subsequent period of slow fermentation are thus seen to represent the velocities of two entirely different chemical reactions.

These considerations also explain why it is the /extra/ carbon dioxide evolved during the initial period, and not the total carbon dioxide, which is equivalent to the added phosphate. As the production of phosphate is proceeding throughout the whole period at a rate which is equivalent to the normal rate of fermentation, it is obviously necessary to deduct the corresponding amount of carbon dioxide from the total evolved in order to ascertain the amount equivalent to the added phosphate.

An explanation is also afforded of the fact that a considerable increase in the concentration of hexosephosphate does not materially increase the normal rate of fermentation. This is probably due to the circumstance that, in accordance with the general behaviour of enzymes in presence of excess of the fermentable substance, the hexosephosphatase hydrolyses approximately equal amounts of hexosephosphate in equal times whatever the concentration of the latter may be, above a certain limit.

According to the experiments of Euler and Johansson [1913] the hydrolytic activity of the hexosephosphatase is greatly diminished by the presence of toluene.

EFFECT OF PHOSPHATE ON THE TOTAL FERMENTATION PRODUCED BY YEAST-JUICE.

The addition of a phosphate to yeast-juice not only produces the effect already described, but also enables a given volume of yeast-juice to effect a larger total fermentation, even after allowance is made for the carbon dioxide equivalent to the quantity of phosphate added. The increase in the case of ordinary yeast-juice has been found to amount to from 10 to 150 per cent. of the original total fermentation [p055] produced by the juice in the absence of added phosphate. The numbers contained in columns 1 and 2 of the table on p. 56 illustrate this effect, the ratio of the total in the presence of phosphate to that obtained in its absence being given, as well as that of the total in presence of phosphate less the equivalent of the phosphate added, to the original fermentation. The cause of this increase in the total fermentation is probably to be sought mainly in a protective action of the excess of hexosephosphate on the various enzymes, for, as has been stated above, the rate of fermentation after the termination of the initial period, is practically the same as in the absence of added phosphate (see p. 43).

Now it follows from equation (1) (p. 51) that in the total absence of phosphate no fermentation should occur, and the experimental realisation of this result would afford very strong evidence in favour of this interpretation of the phenomenon.

Hitherto, however, it has not been found possible to free the materials employed completely from phosphorus compounds which yield phosphates by enzymic hydrolysis during the experiment, but it has been found that when the phosphate contents are reduced to as low a limit as possible, the amount of sugar fermented becomes correspondingly small, and, further, that in these circumstances the addition of a small amount of phosphate or hexosephosphate produces a relatively large increase in the fermenting power of the enzyme.

When the total phosphorus present is thus largely reduced, the increase produced by the addition of a small amount of phosphate may amount to as much as eighty-eight times the original, in addition to the quantity equivalent to the phosphate, whilst the actual total evolved, including this equivalent, may be as much as twenty times the original fermentation. This result must be regarded as strong evidence in favour of the view that phosphates are indispensable for alcoholic fermentation.

The results indicated above were experimentally obtained in three different ways and are exhibited in the following table. In the first place (cols. 3 and 4), advantage was taken of the fact that the residues obtained by filtering yeast-juice through a Martin gelatin filter (p. 59) are sometimes found to be almost free from mineral phosphates, whilst they still contain a small amount of co-enzyme. The experiment then consists in comparing the fermentation produced by such a residue poor in phosphate with that observed when a small amount of phosphate is added. The second method (col. 5) consisted in carrying out two parallel fermentations by means of a residue rendered inactive by filtration [p056] and a solution of co-enzyme free from phosphate and hexosephosphate (p. 67) [Harden and Young, 1910, 2].

The third method (col. 6) consisted in washing zymin with water, to remove soluble phosphates, and then adding to it a solution of co-enzyme containing only a small amount of phosphate, and ascertaining the effect of the addition of a small known amount of hexosephosphate upon the fermentation produced by this mixture [Harden and Young, 1911, 1].

───────────────────────────┬──────┬──────┬──────┬──────┬──────┬─────── │ 1 │ 2 │ 3 │ 4 │ 5 │ 6 ───────────────────────────┼──────┼──────┼──────┼──────┼──────┼─────── │ c.c. │ c.c. │ c.c. │ c.c. │ c.c │ c.c. │ │ │ │ │ │ Gas evolved in absence of │ │ │ │ │ │ added phosphate │ 369 │ 220 │ 1·4 │ 1·2 │ 20·3 │ 1·5 In the presence of │ 629 │ 629 │ 25·8 │ 26·8 │ 92·3 │ 132·7 ───────────────────────────┼──────┼──────┼──────┼──────┼──────┼─────── Increase due to phosphate │ 260 │ 409 │ 24·4 │ 25·6 │ 72·0 │ 131·2 Carbonic acid equivalent │ │ │ │ │ │ to phosphate │ 63 │ 61 │ 16·9 │ 16·8 │ 16·8 │ -- ───────────────────────────┼──────┼──────┼──────┼──────┼──────┼─────── Increase after initial │ │ │ │ │ │ period │ 197 │ 348 │ 7·5 │ 8·8 │ 55·2 │ -- ───────────────────────────┼──────┼──────┼──────┼──────┼──────┼─────── Ratio of totals │ 1·7 │ 2·9 │ 18·4 │ 21·3 │ 4·5 │ 88 Ratio of increase after │ │ │ │ │ │ initial period to │ │ │ │ │ │ original fermentation │ 0·5 │ 1·6 │ 5·3 │ 7·3 │ 2·7 │ -- ───────────────────────────┴──────┴──────┴──────┴──────┴──────┴───────

PRODUCTION OF A FERMENTABLE SUGAR FROM HEXOSEPHOSPHATE BY THE ACTION OF AN ENZYME CONTAINED IN YEAST-JUICE.

The sugar which, according to equation (2) accompanies the phosphate formed by the enzymic hydrolysis of hexosephosphate is under ordinary circumstances fermented by the alcoholic enzyme of the juice and thus escapes detection.

When, however, a solution of a hexosephosphate is exposed to the action of either yeast-juice or zymin, entirely or partially freed from co-enzyme, this sugar, being no longer fermented, accumulates and can be examined. It has thus been found [Harden and Young, 1910, 2] that a sugar is in fact produced in this way which can be fermented by living yeast and exhibits the reactions of fructose, although the presence of other hexoses is not excluded. The products of the enzymic hydrolysis of the hexosephosphates therefore appear to be the same as, or similar to, those formed by the action of acids [Young, 1909].

A further consequence of these facts is that a hexosephosphate will yield carbon dioxide and alcohol when it is added to yeast-juice or zymin, and this has also been found to be the case [Harden and Young, 1910, 2; Iwanoff, 1909, 1]. [p057]

MECHANISM OF THE FORMATION OF HEXOSEDIPHOSPHORIC ACID.

On this subject little is yet known, but a number of extremely interesting results, the interpretation of which is still doubtful, have been obtained by Euler and his colleagues. Euler has obtained a yeast [Yeast H of the St. Erik's brewery in Stockholm] which differs from Munich yeast in several respects. A maceration extract prepared from the yeast dried at 40° in a vacuum produces no effect on a glucose solution containing phosphate. If, however, the glucose solution be previously partially fermented with living yeast and then boiled and filtered, the addition of the extract prepared from Yeast H brings about the esterification of phosphoric acid without any accompanying evolution of carbon dioxide [Euler and Ohlsén, 1911, 1912].

Euler interprets this as follows: (/a/) Glucose itself is not directly esterified, but must first undergo some preliminary change, which is brought about by the action of living yeast. No proof of the existence of a new modification of glucose in this solution has however been advanced, other than its behaviour to extract of Yeast H, so that Euler's conclusion cannot be unreservedly accepted. It is moreover possible and even more probable that some thermostable catalytic substance (perhaps a co-enzyme) passes from the yeast into the glucose solution and enables the yeast extract to attack the glucose and phosphoric acid. A very small degree of esterification was also produced when an extract having no action on glucose and phosphate was added to glucose which had been treated with 2 per cent. caustic soda for forty hours, but the nature of the compound formed was not ascertained [Euler and Johansson, 1912, 4]. (/b/) The esterification of phosphoric acid without the evolution of carbon dioxide implies that the enzyme by which this process is effected is distinct from that which causes the actual decomposition of the sugar. Euler goes further than this and regards the enzyme as a purely synthetic one, giving it the name of hexosephosphatese to distinguish it from the hexosephosphatase which hydrolyses the hexosephosphate.

The evidence on which this conclusion is based cannot be regarded as satisfactory, inasmuch as it consists in the observation that /in presence of sugar/ yeast extract does not hydrolyse the phosphoric ester. This, however, could not be expected since hydrolysis and synthesis under these conditions would ultimately proceed at equal rates.

In any case the adoption of this nomenclature is inconsistent with the conception of an enzyme as a catalyst and is therefore inadvisable until the reaction has been much more thoroughly studied. [p058]

It may further be pointed out that no proof has yet been advanced that the phosphoric ester produced without evolution of carbon dioxide is identical with hexosediphosphoric acid produced with evolution of carbon dioxide. It is by no means improbable that it represents some intermediate stage in the production of the latter (see p. 117).

Euler's other results on this subject may be briefly summarised as follows:--

(1) In presence of excess of sugar the esterification of the phosphoric acid proceeds by a monomolecular reaction and is most rapid in faintly alkaline reaction [Euler and Kullberg, 1911, 3].

(2) When yeast extract has been heated for 30 minutes to 40° it effects the esterification of phosphoric acid at a much greater rate than the unheated extract (2-10 times). Heating at 50° for 30 minutes however completely inactivates the extract. The cause of the activation is as yet unknown. The temperature coefficient for the unheated extract (17·5°-30°) is 1·4-1·5 for 10° rise of temperature [Euler and Ohlsén, 1911].

(3) Yeasts which in the dried state all produce rapid esterification of phosphoric acid, yield extracts of very unequal powers in this respect [Euler, 1912, 1]. [p059]