HYDROLYSIS OF SUCROSE BY INVERTASE IN THE PRES- ENCE OF a-METHYL GLUCOSIDE. II.*

BY J. M. NELSON AND C. IRWIN POST.

(From the Department of Chemistry, Columbia University, New York.)

(Received for publication, March 23, 1926.)

It has been pointed out in previous papers from this laboratory that the hydrolysis of a 10 per cent sucrose solution by invertase, obtained from a brewery yeast, can be represented by the empirical equation (1)

N = 5 100

log ____ 100 - + 0.002642 p - 0.00000886 p

p* - 0.0000001034 p3 1 (11

in which N remains constant throughout any one hydrolysis (if the latter is not deviated from its normal course by the inactiva- tion of the enzyme or for some other reason) (see Table I A). The quantities p and t represent the per cent sucrose hydrolyzed and time respectively.

Presence of Added Invert Sugar Modijies the Course of Hydroly- .&-.---When invert sugar is added to the 10 per cent sucrose solu- tion the hydrolysis is retarded. By applying equation (1) to the data obtained from such a hydrolysis, Table I B, it will be noticed that the values for the parameter N are no longer constant but decrease as the reaction proceeds. Since N may be looked upon as a measure of the velocity of hydrolysis, this decrease shows that not only is the reaction slowed up as if less invertase had been used, but the rate becomes less and less as the hydrolysis progresses.

InJluence of Invert Sugar Is Opposite in Character to That of a-Methyl Glucoside:-In the first paper of this series, Nelson and Freeman (2) pointed out that, although a-methyl glucoside retards

* Published as Contribution No. 511 from the Department of Chem- istry, Columbia University, New York.

265

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266 Hydrolysis of Sucrose by Invertase. II

the hydrolysis of sucrose by invertase, still, when equation (1) is applied to the results obtained from the hydrolysis of a 10 per zcent sucrose solution containing glucoside, the calculated values for N show an increase as the reaction proceeds (Table I C). This increase in N is just the opposite to the decrease in N in the case

TABLE I.

pH 4.67. Temperature 25°C.

A. 10 per cent sucrose.

Time. Rotation. ______

min.

0 22.30' 15 18.70 30 15.20 45 11.95 0 9.00 80 5.51

110 1.40 co -6.60

Hydrol- ysi s. N

per cent

12.46 599 24.57 601 35.81 602 46.02 601 58.10 601 73.32 603

C. 10 per cent sucrose + 0.125 gm. of

glucoside.

0 22.90 7.5 21.70

15 20.49 45 15.71 80 10.50

120 5.32 150 2.13

m -6.00

4.15 8.34

24.88 42.91 60.83 71.87

395 407 416 426 438

r 1

-

-

-I-

Time. Rotation. Hydrol- ysis. N

-- min. per cent

0 18.40” 7.5 17.04 4.71 447

15 15.67 9.45 444 45 10.58 27.06 435 80 5.50 44.63 425

115 1.42 58.75 416 150 -1.75 69.72 416

cc -10.50

D. 10 per cent sucrose + 0.125 gm. of

glucoside + 6 gm. of

B. 0 per cent sucrose + 6 gm. of in-

vert sugar.

invert sugar, T

0 19.08 15 17.08 60 11.38

105 6.20 140 2.98 180 -0.43

co -9.82

6.92 327 26.66 328 44.57 331 56.07 328 67.51 330

-

where invert sugar has been added to the hydrolyzing sucrose solution. In other words, when the glucoside is the retardant, then (if N is taken as a measure of the rate of hydrolysis) its retarding influence becomes less and less as the reaction progresses, while when added invert sugar acts as the retardant, its retarding influence increases as the reaction continues.

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J. M. Nelson and C. I. Post 267

Relative Amounts of Glucoside and Invert Sugar Required to OfseE Each Other’s In$uence.--Since the two retardants, glucoside and invert sugar, affect the hydrolysis in different ways, it was thought that very likely if these retardants were present in certain definite amounts with respect to each other, then the course of the reac- tion might again tend to become normal. The course of a hydroly- sis is considered as normal, when equation (1) gives constant values for N, or when the per cent hydrolyzed-time curve (like those in Fig. 1) is similar in shape to a corresponding curve representing the hydrolysis of a 10 per cent sucrose solution containing no added

80 I

0 i-/ME - MINUTES

FIG. 1.

retardant. When a hydrolysis curve can be made superimpos- able upon another such curve by multiplying the time coordinates by some number, then the two curves are considered to be similar in shape.

By a series of trial mixtures of invert sugar and glucoside, it was found that when the two retardants were present in amounts of 6 gm. and 0.125 gm. per 100 cc., respectively, then the course of the hydrolysis tended to become normal as shown by the values for N in Table I D. And it was also found that when only 3 gm. of invert sugar were added per 100 cc. then only half as much, or

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TABLE II. -

_

-

1

A. 10 per cent sucrose + 0.0625 gm. of

glucoside + 3 gm. of invert sugar.

pH 4.64.

B. LO per cent sucrose + 0.125 gm. of

glucoside + 6 gm. of invert sugar.

pH 2.98. -

.-

-

Hydrol- ysis.

Hydrol- ysis. N Time. Rotation.

min.

0 10 20 30 40 55 70 m

24.48’ 20.15 16.00 12.12

8.58 4.00 0.42

-9.32

C.

N Time. Rotation.

min.

0 10 20 40 60 80

100 co

-

-

22.38’ 19.24 16.15 10.42

5.40 1.16

-2.24 -11.42

--

-

per cent

12.81 918 25.09 922 36.57 924 47.04 924 60.59 925 71.18 923

per cent

9.29 18.43 35.38 50.24 62.78 72.84

661 667 668 667 668 671

D. 10 per cent sucrose + 0.125 gm. of

glucoside + 6 gm. of invert sugar.

pH 4.64.

10 per cent sucrose + 0.125 gm. of glucoside + 6 gm. of invert

sugar. pH 6.73.

-

-

0 22.57 20 17.72 40 13.15 61 8.73 80 5.16

100 1.91 130 -2.05

m -11.23

0 22.31 22.5 15.94 46.5 9.77

101.75 -1.13 117.17 -3.08

m -11.49

F.

14.35 514 27.87 516 40.95 516 51.51 516 61.12 515 72.84 517

18.85 37.10 69.35 75.12

608 606 609 605

2 per sucrose f 0.125 gm. of glucoside + 6 gm. of invert

sugar. pH 4.65.

E. pH 4.65. 2 per cent sucrose.

-

-

I I 0 4.46

10 3.40 20 2.44 32 1.46 40 0.94 55 0.16 90 -0.80 co -1.32

0 1.39 10 0.45 16.26 20 -0.37 30.45 30 -1.08 42.73 40 -1.70 53.46 55 -2.43 66.09

100 -3.69 87.89 co -6.44

18.34 34.95 51.90 60.90 74.39 91.00

- 268

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J. M. Nelson and C. I. Post

TABIS II-Conchded.

G. pH 4.65. 5 per cent sucrose.

E

Time.

min.

0 10 20 30 40 55 95 m

iotation.

11.13’ 8.64 6.33 4.29 2.56 0.52

-2.20 -3.32

Hydrol- ysis.

per cent

17.26 33.22 47.34 59.32 73.43 92.25

N Time. Rotation.

H. i per cent sucrose + 0.125 gm. of

glucoside + 6 gm. of invert sugar.

pH 4.65.

min.

0 20 40 60 80

100 180

03

8.01" 5.53 3.27 1.29

-0.41 -1.79 -4.95 -6.44

Hydrol- ysis.

per cent

17.16 32.80 46.51 58.27 67.82 89.70

N

0.0625 gm., glucoside was required to make the hydrolysis proceed along the curve followed when the retardants are absent (see Table II A). It was also found that this ratio of the two retard- ants was independent of the hydrogen ion concentration and of the concentration of the invertase causing the hydrolysis. The last two statements are borne out by the experimental data given in Table II B, C, and Di

To see whether the ratio between the amounts of the two retard- ants present was independent of the sucrose concentration, hydrol- yses were run on 2 and 5 per cent sucrose solutions besides the 10 per cent mentioned above, both with and without the presence of retardants (see Table II E, F, G, and H). Since the equation of Nelson and Hitchcock is not applicable to sucrose solutions of these concentrations, a graphical method had to be used instead. This method consisted in plotting the per cent hydrolyzed as ordinates against the corresponding time as abscissa. When the time coordinates of the curves obtained in this way, representing hydrolyses in the presence of the retardants, were multiplied by a given number, then it was found that these curves become super- imposable upon the corresponding curves representing hydrolyses identical in all respects except that the retardants were absent. This shows that the above ratio of glucoside to invert sugar tends

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270 Hydrolysis of Sucrose by Invertase. II

to give a normal hydrolysis curve with sucrose concentrations varying from 2 gm. to 10 gm. per 100 cc. of solution.

Glucoside and Invert Sugar Neutralize Each Other’s Injluence on Relationship between Sucrose Concentration and Rate of Hydrolysis. -Nelson and Freeman also pointed out that the relation between sucrose concentration and rate of hydrolysis was affected by the presence of a-methyl glucoside. They showed this in the follow- ing way. Several solutions containing different amounts of SU-

crose were hydrolyzed under the same conditions of temperature, hydrogen ion concentration, and amount of invertase. In order to ascertain the relative initial rates of hydrolysis of these various

3 0 / 2 3 4 5 6 7 8 9 /O II 12

GRAMS SUCROSE PER IO0 CC.

FIG. 2.

sucrose solutions, the results obtained in each individual hydrolysis were plotted as per cent hydrolyzed against time, and tangents drawn to the origin of these hydrolysis curves. The values of these tangents were then taken as the relative initial rates of hydrolysis of their respective solutions. This relationship be- tween the initial velocities and the concentration of sucrose is shown graphically by Curve B in Fig. 2. Curve A in the same figure was obtained in the same way as that used for Curve B, except each of the various sucrose solutions contained a constant amount, 1 per cent, of added glucoside. For the sake of compari- son of the initial rates of hydrolysis, the maximum rates in each

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J. M. Nelson and C. I. Post 271

set of experiments, i.e. the hydrolysis in the absence and presence of glucoside, were put equal to 1000 and the lesser rates as frac- tions thereof. In other words, the change in velocity with respect to the change in sucrose concentration in the two cases was put on the same relative scale. By comparing the shapes of the two curves, A and B in Fig. 2, it will be seen that the retarda- tion of the hydrolysis due to the presence of a constant amount of a-methyl glucoside increases as the sucrose concentration increases,

TABLE III.

-4. pII 4.65. Temper&n-e 25°C.

SUCKW. Initial velocity. Relative velocity.

per cent

1 0.067, 604 2 0.093 838 3 0.106 955 4 0.1097 988 4.25 0.110 991 5 0.111 1000 6 0.111 1000 7 0.111 1000

B. Hydrolyses in presence of 6 per cent invert sugar.

1 0.048 380 2 0.076 605 3 0.094 755 4 0.106 850 5 0.114 915 6 0.117 935 8 0.121 970

10 0.12<5 1000

or the retardation is least in the more dilute sucrose solutions and reaches a maximum when the sucrose concentration has reached a value corresponding to a maximum rate of hydrolysis.

To see whether invert sugar influences the relation between the rate of hydrolysis and the concentration of sucrose in the opposite way from that of glucoside, a series of hydrolyses was run similar to the series run by Nelson and Freeman for Curve A in Fig. 2, except that 6 per cent of invert sugar was added to each of the different sucrose solutions instead of 1 per cent of glucoside. The

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272 Hydrolysis of Sucrose by Invertase. II

relative initial rates of hydrolysis obtained from this set of hydroly- ses, Table III B, have been plotted, giving Curve C in Fig. 2. By comparing the shape of Curve C with those of -4 and B in the same figure, it becomes evident that the invert sugar affects the relation between the rate of hydrolyses and sucrose concentration in a different manner from the effect due to glucoside. The invert sugar exerts its greatest retardation in the dilute sucrose solutions, which is just the opposite effect to that observed in the case when glucoside is the retardant.

It has been shown above that when the relative amounts of glucoside and invert sugar added to a 10 per cent sucrose solution undergoing hydrolysis bear a certain ratio with respect to one another, then the course of the hydrolysis tends to become normal; i.e., when the results from the hydrolyses are substituted in equa- tion (l), practically constant values for N are obtained. When this same ratio of glucoside and invert sugar was used to study their combined effect on the relationship between the rate of hy- drolysis and concentration of sucrose, it was found that here again the two retardants in these proportions offset each other’s influence. The procedure followed in these experiments was the same as that used in determining the relative initial velocities when the influence of each retardant on the relation between the ve- locity and sucrose concentration was studied separately. The results obtained from these experiments, i.e. the initial rates of hydrolysis when the mixt,ure of the two retardants was used, are given in Table IV. If these values are plotted similarly to the results represented by curves in Fig. 2, it will be found that the shape of the resulting curve coincides with Curve B in that figure. It will be recalled that Curve B represents the change in the initial rate of hydrolysis for increasing sucrose concentration in the absence of retardants. Since the curve representing the change in initial rate wit,h increasing sucrose concentration is superimposable upon Curve B, this means that when the two retardants are both pres- ent in the hydrolyzing sucrose solution they exert a peculiar neutralizing effect upon one another.

E$ect of Invert Sugar on the pH Optimum Curve.-Nelson and Freeman examined the influence of a-methyl glucoside on the relation between hydrogen ion concentration and rate of hydrolysis of sucrose by invertase from brewers’ yeast. This was done by

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J. M. Nelson and C. I. Post 273

TABLE IV.

Hydrolyses in the presence of 6 ner cent invert sugar and 0.125 per cent

SUWOS‘S.

\ per cent

1 2 3 4 5 6 8

a-methyl glucoside.

Initial velocity. Relative velocity.

0.0187 593 0.0263 838 0.0296 940 0.0318 1000 0.0313 1000 0.0314 1000 0.0316 1000

TABLE V.

A. 6 per cent sucrose. Invertase BW. Temperature 25°C.

PH Initial velocity. Relative velocity.

2.90 0 143 877 3.15 0.148 908 3.75 0.156 957 4.21 0.160 982 4.64 0.163 1000 5.27 0.161 988 5.99 0.144 883 6.31 0.134 822 6.70 0.111 681 6.78 0.105 644

B. 6 per cent sucrose and 6 per cent invert sugar. Invertase BW. Temperature 25°C.

2.80 0.100 855 3.06 0.104 889 3.65 0.111 949 4.17 0.114 975 4.64 0.117 1000 5.24 0.114 975 5.95 0.103 880 6.31 0.094 803 6.70 0.083 710 6.79 0.080 684

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274 Hydrolysis of Sucrose by Invertase. II

determining the initial rates of hydrolysis of several 10 percent sucrose solutions, all containing 1 per cent glucoside, at different hydrogen ion concentrations, and then plotting the values thus obtained against their corresponding hydrogen ion concentrations. This curve was then compared with the curve, the data for which were obtained in the same way except that retardant was ab-

0.17

0.16

0. I5

PH

A-bAL6% SUCROSE B-88’6% ” +6X

INVERT SUGAR

600

234567

PH FIG. 3.

sent, and it was found that by multiplying the ordinates of one curve by a suitable constant the two curves then became superimposable.

In the present study the influence of invert sugar on the opti- mum pH curve has been investigated. The initial rates of hydrolyses, determined as described above, were run on 6 per cent

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J. M. Nelson and C. I. Post 275

sucrose solutions at various hydrogen ion concentrations, both with and without 6 per cent added invert sugar. The results obtained are given in Table V A and B, and are shown graphically in li’ig. 3. It will be seen that the optimum point is unchanged, i.e. it still remains at pH 4.5 to 4.7, and that here again the two curves, in the presence and absence of retardant, may be made superimposable by multiplying the ordinates of one by a suitable constant.

It would appear, then, that invert sugar and a-methyl glucoside have at least one point of similarity, in that their retardation is not affected by the acidity of the solution, nor does either one change the optimum point of the hydrolysis of sucrose by inver- tase at these concentrations.

SUMMARY.

1. It has been found that, when a-methyl glucoside and invert sugar are present in the ratio of 0.125 to 6 per cent, the hydrolysis of sucrose by yeast invertase tends to follow the same course as when the two retardants are absent, and that the effect of the mixture is the same as that of using less concentration of the enzyme.

2. It has been found that invert sugar and a-methyl glucoside tend to neutralize each other’s influence on the relationship be- tween rate of hydrolysis and sucrose concentration.

3. It has been found that retardation due to invert sugar, like that due to a-methyl glucoside is independent of the hydrogen ion concentration.

BIBLIOGRAPHY.

1. Nelson, J. M., and Hitchcock, D. I., J. Am. Chem. Sot., 1921, xliii, 2632. 2. Nelson, J. M., and Freeman, B., J. Biol. Chem., 1925, lxiii, 365.

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J. M. Nelson and C. Irwin Post-METHYL GLUCOSIDE. II

αINVERTASE IN THE PRESENCE OF HYDROLYSIS OF SUCROSE BY

1926, 68:265-275.J. Biol. Chem. 

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