THE KINETICS O F CATALYZED SUGAR HYDROLYSIS AS A FUNCTION O F TEMPERATURE

I R W I N W SIZER . Department of Biology and Public Health, Mussacliuoetts I n s t i t u t e of Technology, Cambridge

FOUR FIGURES

The velocity of most chemical reactions increases exponentially with temperature. The data conform with considerable accuracy t o the Arrhenins equation which is written conveniently in the form

where k, and k are the rates of reaction at the absolute , temperatures TI and T,, R is the gas constant, and p is the temperature characteristic representing the activation energy in calories of the reaction (Glasstone, '33). Although at first only a n empirical relationship, this equation has since been amply confirmed from theoretical physics and chemistry (Crozier and Hoagland, '34). The temperature characteristic, or p value, is considered t o represent the energy in calories required to activate 1gm. mol of the catalyst for the reaction. If this is true, different reactions having the same catalyst will have the same temperature characteristic. This has been experimentally verified for certain inorganic catalysts. The same postulates for the influence of temperature on reaction velocity would be expected to apply to biochemical reactions catalyzed by enzymes. The p value, or critical increment, for such a reaction should be constant over the whole range of temperature until the inactivation temperatnre of the enzyme is reached. The absolute velocity of the reaction will be altered of course, when certain properties of
61

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IRWIN W. SIZER

the medium are changed, for example the (H+) of the solution, the electrolyte concentration, and the relative amounts of substrate and enzyme. The temperature characteristic should be independent of such environmental changes, however, unless these alterations have modified the physical and chemical structure of the enzyme. If p characterizes the catalyst, and not the reaction, then the same temperature characteristic should be obtained when a single enzyme acts on different substrates. Conversely, enzymes which are chemically distinct should yield different p values when acting on the same substrate. Previous workers have found that the tcmperature characteristic for most enzyme reactions is not a constant, but decreases with rise in temperature. F o r yeast invertase ~1 was found t o decrease linearly with temperature according t o the equation proposed by Vosburgh (Nelson and Bloomfield, '24) : p = 12,300 - 117t. Nelson and Bloomfield, using the temperatures 25, 30 and 35"C., found that p decreased rapidly with rise in (H+) and temperature, but was relatively constant in the region of pH for optimum activity ( p H 4.7). Auden and Dawson ( '31) using concentrated sugar solutions and high temperatures also reported a decline of p with temperature. Kertesz ('33) found that p declined not only at temperatures above 0C. but at temperatures below this point as well. The above results are all contrary to what would be expected on theoretical grounds. According to some (Raldane, '30) this fact throws doubt on all calculations of critical increments f o r biological processes, since a biological system is assumed t o be more complex than a simple enzyme reaction. Because of the discrepancy between expe1:imental results and theory, this study of catalyzed cane sugar hydrolysis as a function of temperature was undertaken.
METHOD

Sugar solutions were placed in a 200-mm. polariscope tube and the course of the reaction followed by means of a Schmidt and Haensch polarimeter. - Mazda 60-watt blue bulb was 9 used throughout as the source of illumination. The tempera-

KINETICS O F CATALYZED SUGAR HYDROLYSIS

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ture inside the tube was controlled to 2 0.1C. by means of a water thermostat. T a p water passed through coils in this bath and then through the water jacket of the polariscope tube. The temperature was measured a t the outlet from this jacket and was found to coincide with that of the solution inside the tube. I n all cases 10 cc. of the sugar solution and 2.5 cc. of the enzyme solution were used. These solutions were adjusted to the desired temperature and then thoroughly mixed before being placed in the polariscope tube. To avoid temperature inactivation, the enzyme solution W R S allowed only 2 minutes adaptation at the desired temperature before being used. The rotation of the solution was measured at frequent intervals during the course of the reaction. The interval between readings was determined by the rate of hydrolysis. A t the end of each inversion the pH of the solution was measured by means of the quinhydrone electrode. To prevent bacterial contamination 2 drops of toluene were added to the stock sugar and enzyme solutions. Only commercial enzyme preparations were used i n these experiments. Wallersteins yeast invertase scales were employed, one preparation (WRL) containing melibiase a s a n impurity, the other ( W B L ) purified free from melibiase. Experiments were also performed using Difco liquid yeast invertase solution. I1IercBs malt diastase was studied with the purpose of comparing it with yeast invertase. Enzyme solutions were kept on ice, and no change in activity was observed over a period of several months.
RESULTS

Iwversion of stm*ose by HGl Inversion of 10% sucrose by 0.5N HC1 was studied at seven temperatures between 5 and 50C. This experiment was r u n as a means of standardizing technique and also with the intent of comparing the bio-catalysts with a n inorganic one. Sugar inversion catalyzed by the hydrogen ion follows the monomolecular reaction equation
1 k = -log t -a a-x

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IRWIN W. SIZEP,

over the entire course of hydrolysis. Log k plotted against 1/T yields a linear relationship; the slope of the line drawn through the plotted points corresponds to a j.~ = 26,000. This agrees well with the original value of 25,600 obtained by Arrhenius (1889).

Inversion of sucrose by yeast invertase A solution containing 16% sucrose and 0.2% invertase (WBL) was used. This solution was buffered to pH 5.02 with

I3

'9

es

1.1

*
0.9

e
I6

l
24

MINUTE^

Fig.1 Hydrolysis of a 16% solution of sucrose, buffered t o pH 5.02 with NaH,PO,, catalyzed by 0.2% yeast iiivertase (WBL). Log rotatioii (R, - R m ) of the solution is plotted against elapsed time in minutes. Inversion a t 4.2"C.- 0; i50-x;2 5 0 - 0 ; 3 5 0 - e ; 4 0 - + ; 5 0 0 - a > .

NaH,PO,. The course of inversion was followed a t six different temperatures and the data analyzed in accordance with the monomolecular equation. In figure 1 log rotation (R, - R,) is plotted against elapsed time in minutes for the data obtained on inversion at the various temperatures. It is obvious from the figure that over the major portion of the reaction (the log phase) a straight line can be drawn through the plotted points; this is equivalent to saying that the hydrolysis follows the monomolecular equation. It deviates sharply from this at the beginning of the reaction (the lag

KINETICS O F CATALYZED SUGAIl HYDROLYSIS

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phase) and again toward the end (the inhibition phase). The lower the temperature the longer the lag phase, the longer is the log period, and the later the inhibition period sets in. I n terms of the per cent of sugar hydrolyzed, however, the duration of each of the three phases of the reaction is not altered by change in temperature. The monomolecular velocity constants were calculated for the log phase of the reaction for the six different temperatures (table 1). Log k was then plotted against 1/T (see plain
TABLE 1

Uninioleeular vetocity constants for the inwemion of 16% sucrose 6y 0.2% invertase ( W B L )
~~ ~ ~ ~~

____~

__
-

1000 k
TEMPERATURE

pH 3.24
.~
~~~

pH 5 02
~~~

pH 7.94

C.

3.6 3.8 4.2 4.4 14 15 25 27 35 38.1

40
-

0.777 7.89 4.63 4.16 17.8 10.0 20.0 33.7 11.1 03.0 42.4 106
1
I
1

4.38 10.8 21.1 37.4 1.73 2.56 2.41

50

'4%

-_ sucrose used instead of 16%.

50.8 78.0
~~ ~~ ~

circles in fig. 2). straight line may be drawn through the plotted points; its slope corresponds to a p value of 12,000. The point for 50C. appears to he lower than the expected value; this is probably due to the heat inactivation of the enzyme, a factor playing an important role in slowing down the hydrolysis a t this high temperature. I n contrast with the work of others the thermal increment was found t o have a constant value of 12,000 over the temperature range from 4 t o 40C. This applies only to that portion of the inversion which follows the unimolccular equation. During the last
J O U H N A L O W CBLLUL.4R AND COIIIPARATIT'E PHYSIOI.OCY,

\ O L , 10, NO.

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IRWIN W. SIZER

stage of the reaction p will probably vary with the temperature and other factors, The effect on the kinetics of inversion, of electrolytes, changes in (H+), and change in sugar concentration was next investigated. Using the same sugar and enzyme concentration as before the following solutions were used; a solution of pH 3.24, HCl furnishing the acidity; a solution of pH 5.82 adding no electrolyte; a solution of pH 7.94, a NaH,PO,-KOH buffer supplying the necessary alkalinity.

1.5
n

2
x

x
rl 0 1.0
0

Fig. 2 Log of the monomolecular velocity constants, f o r inversion in a solution containing 16% sucrose and 0.2% yeast invertase ( m L ) plotted against 1/T. Solution buffered t o p H 5.02 with NaH,PO, - 0; solution unbuffered, p H 5.86 - X ; solution adjusted to p H 3.24 with HCI - 0 ; solution buffered to pH 7.94 with NaH,PO, -KOH a 4% sucrose solution buffered to p H 5.38 - 8 . The points for temperatures below 4orO, except those for pH 7.94, fall along the straight line of slope corresponding to p = 12,000.

+,

Using a solution buffered to pH 5.38 with NaH,PO, inversion was studied using only a 4% sugar solution instead of the usual 16% (as before the solution contained 0.2% invertase). The results of the several different experiments are summarized graphically in figure 2. The velocity constants have all been multiplied by factors so that the ordinates in the log k vs. 1/T graph would be similar to those for inversion a t pH 5.02. It is evident from the graph that for the monomolecular phase of the reaction the critical increment is independent of pH changes from 3.2 to 7.9, of sucrose changes

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from 16 to 4%, aiid is unaltered by the presence of various electrolytes. The points f o r pH 7.94 deserve some comment. I t is only at temperatures below 20C. that the points fit the curve ; above this temperature they fall off below the expected values. At 35C. the velocity of inversion is even slower than a t 25C. Apparently in the presence of alkali, yeast invertase i s very unstable even a t ordinary temperatures.
TABLE 2

Unimolecular velocity constants for the inversion of 16% sucrose by 0.2% invertme ( W R L )
1000 k
FEMPEBATURE
_~_____.

"0.

_____

pH 3.22

pH4.74

pH 4 . 9 8 1

pH 5.63

3.7 4.2 4.4 5.73 10 14 15 20 25 27 30 35 37.9 40 45 50

3.10
5.00

4.49 6.13 8.50 7.16 10.7 21.1 11.8 16.8 25.8 19.6 36.4 36.1 52.2 36.0 61.5 72.9 89.6 53.1 80.4 41.2 11.9 24.0

' 0.1%

invertase used instead of 0.2%.

A similar series of cxperirnents was run with a different yeast invertase (TVRL) preparation using the pH's, 3.22, 4.74 and 5.63, with the same buffers as before (table 2). I n addition a 16% sugar solution ( p H =4.94) containing oiily 0.1% invertase instead of the usual 0.2% was studied. All velocity constants were multiplied by a factor so that they would be comparable with those for p H 4.74. It is evident from figure 3 (lower curve) that all the points lie along the straight line whose slope corresponds to the value of 12,000.

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I R W I N W. SIZER

Again a t 50C. the points fall off from the line probably because of the inactivation of the invertase at this high temperature. It is evident that WRL invertase gives the same temperature characteristic as WBL invertase, and that this value is likewise independent of changes in the medium.

Fig. 3 Log of the monomolecular velocity constants, for the hydrolysis of sucrose and raffinose catalyzed by yeast invertase, plotted against 1/T. Upper curve, sucrose inversion catalyzed by 4% Difco yeast invertase. 40% sucrose buffered with NaH,PO, t o pH 5.86 - 0.Sixteen per cent sucrose buffered t o pH 5.32 with NaI,PO, - X. The slope of the curve corresponds t o a p = 12,500. M i d d l e curve, hydrolysis of an S70 raffiuose solution buffered t o pH 5.0 with NaH,PO,, catalyzed by 0.2% yeast invertase (WBL). p = 12,200. Lower curve, inversion of 16% sucrose catalyzed by 0.2% yeast invertase unbuffered, pH 5.63 - X ; (WRL). Buffered to pH 4.74 with NaH,PO, - 0; HCI added, pH 3.22 buffered t o p H 4.94 with NaH,PO, but with only 0.1% invertase iustead of 0.2% - 8. A t temperatures below 45C. p = 12,000.

-+;

A third series of determinations was made, this time using sugar solutions containing 476 Difco yeast invertase (table 3). I n the first group of experiments the solution contained 16% sucrose and was buffered t o p H 5.32 with NaH,PO,; in the second the solution contained 40% sucrose (40 gm. per 100 cc. solution) and was buffered t o p H 5.86 with NaH,P04. Before

KINETICS OF CATALYZED SUGAR HYDROLYSIS

69

plotting the results the velocity constants for the 40% sucrose solutions were multiplied by a constant to make them comparable to those for 16% sucrose. A glance at figure 3 (upper curve) reveals the fact that the p value has roughly the value of 12,500 over tlie temperature range from 3.4 to 38C. for both sugar concentrations. This value is probably not significantly different from that of 12,000 obtained for the
TABLE 3

Unimolecular veloczty constants f o r the hydrolysis of sucrose by znvertase ( D i f c o ) and malt dzastase, and of r a f i n o s e by invertase ( W B L )
1000 k
~~ ~

TEMPERATURE

~00.

16% sucrose 4 % invertase ( D . ) pH 5.32

40 70sucrose 4 % invertase ( D ) pH 5 . 8 6
-

3 % raffinose 0 2 % invertase (WBL) pH 4.99

16% sucrose 1% malt diastase pH 4.75

2.7 3.4 3.8 4 8.2 10.9 14 15 17 23.5 25 86 28 34 35 36 38 40 45

0.0297 4.44
0.992

3.72 0.0463 0.0522 10.8 2.78 8.77 0.0847 0.151

17.9

24.9

6.64 0.208 0.314 34.4

4. 80
15.4 0.373 0.427

other two yeast invertases. Thus the p value for the monomolecular phase of the reaction is the same for different samples of yeast invcrtase of various degrees of purity, is independent of the concentration of enzyme or substrate, is unaltered by changes in pH from 3.2 to 7.9 and is not affected by the presence of electrolytes. It must be remembered that a change in any one of these factors will modify the velocity of inversion at any one temperature.

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IRWIN W. SIZER

Inversioiz of rafiizose by. yenst invertase

The temperature characteristic is believed to represent the energy of activation of the catalyst i n a chemical reaction. This can be tested by using the same enzyme on different substrates and comparing the p values obtained. A search was made for another substrate for yeast invertase. It was inert toward maltose, lactose, and melizitose, but did hydrolyze raffinose (about four times as slowly a s sucrose) to fructose and melibiose. Yeast invertase thus appears to be a fructosidase requiring a substrate with the fructose radical at one end of the molecule (Haldane, '30). The solution employed contained 8% raffinose, buffered to pH 5.0 with NaH,PO,, and 0.2% yeast invertase (WRL containing no melibiase). The method of analyzing the results was the same a s for the sucrose experiments (table 3). As before, the curves in the monomolecular plots can be divided into three sections, the lag, log and inhibition periods. As usual, the velocity constants were calculated from the log phases. It is apparent from figure 3 (middle curve) that p is constant over the temperature range from 4 to 35C. and has the value of 12,200. It becomes evident, then, that the same critical increment is obtained whether yeast invertase catalyzes the hydrolysis of sucrose or of raffinose. These results also indicate that in yeast invertase there are not two enzymes, one specific for sucrose, the other for raffinose, but that the same enzyme acts on both substrates. The identity of these two enzymes has been contested, but at present the bulk of the evidence from other sources also indicates that the same enzyme acts on both these fructosides (Nelson, '33).

Inversion of szccyose b y malt invertuse

Most of the evidence available is in accord with the view that enzymes from different sources which act upon a given substrate are not identical. F o r example, such enzymes may have different degrees of specificity, different pH optima, and different heat inactivation temperatures (Nelson, '33). It

K I N E T I C S O F CATALYZED SUGAR HYDROLYSIS

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was a problem of considerable interest to determine whether invertase from different sources would have the same temperature characteristic as yeast invertase. The diastase preparation from malt was found to have a mild effect on sucrose inversion and was employed in this study. Since the enzyme was only slightly water soluble, a 5% suspension of the enzyme was made. The solution was then filtered free of the insoluble material; 2.5 cc. of the filtrate was added to 10 cc. of the sucrose solution. This enzyme preparation is less specific than yeast invertase for it hydrolyzes maltose and

Fig. 4 Log of the monomolecular velocity constants f o r the hydrolysis of 16% sucrose by 1% malt invertase plotted against 1/T. The solution was buffered to pH 4.75 with NaH,PO,. P = 13,000. The points fall off from the straight line above 34C.

melizitose in addition to sucrose and rafbosc. It also has a lower heat inactivation temperature than yeast invertase. Inversion was studied using a 16% sucrose solution buffered with NaH,P04 to pH 4.75. The results were analyzed by the same method as in previous experiments (table 3 ) . I n contrast to the results with yeast invertase the reaction course was monomolecular from the beginning; no lag phase was observed. Inversion was, however, about 300 times slower than with yeast invertase. The inhibition phase was initiated after about 30% of the inversion was complete. When the logarithm of the monomolecular velocity constant is plotted

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IRWIN W. SIZER

against 1/T (fig. 4) it is clear that a straight line may be drawn through the points over thc temperature rangc from 2.8 to 35C. Above 35C. the malt invertase undergoes heat inactivation. The slope of the line corresponds to a p of 13,000. The difference in the course of inversion, in the temperature of heat inactivation, and in the critical increments, all indicate that malt invertase, yeast invertase, and HCl are chcmically distinct, and behave diflercntly in catalyzing sucrose hydrolysis.
DISCUSSION

Aaalysis of data
Velocity constants were calculated from the early portion of the hydrolysis where the reaction appeared to be unimolecular. This phase of the reaction is not necessarily more typical of enzymic sugar hydrolysis than is the last phase (inhibitory), where the velocity is appreciably decreased. Most workers who have studied the kinetics of enzyme reactions, however, emphasize the importance of the early portion of the reaction for a quantitative study of the experimental data (Tauber, ( '37). Methods other than that of calculating unimolecular velocity constants may be employed f o r determining rate of sugar hydrolysis at the various temperatures. The duration of the lag phase in the unimolecular plot of the data, or of the time elapsed bcforc the onset of the inhibition phase, are equally good measures of reaction velocity. Another criterion of reaction rate is the time required to effect a given per cent hydrolysis. The velocity of inversion may also be calculated from the slope of the linear portion of the curve, when rotation is plotted against time (compare Nelson and Bloomfield, '24; also White, '33), or in the bimolecular plot of the data. An analysis of the data obtained by these methods also indicate a constant p of 12,000 in the Arrhenius equation. It should be pointed out, however, that these methods likewise apply only to data for the first two-thirds of the hydrolysis.

IIINETICS OF CATALYZED SUGBEl HYDROLYSIS

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All the workers on invertase have calculated p by substituting values in the Arrhenius equation. A much more satisfactory method is to plot log rate of inversion against 1 / T and calculate p from the slope of the line drawn through the points. A recalculation of some of the data in the literature shows that a decrease in p with rise in temperature is by no means always obtained. The data of Euler and Laurin ('19-'20, '20) and Euler and Ugglas ('lo) indicate that p is fairly constant over the temperature range from 0 to 20C. at about 11,400. A re-analysis of the work of Auden and Dawson ('31) on 70% and 55% sucrose indicates a constant p of about 11,000 over the range from 38 to 57C. The data of O'Sullivan and Thompson (1890) and Tammann (1892) do not lend themselves to this interpretation. It is interesting to note that in both formulae proposed t o express the change of p with temperature (Euler and Laurin, '20; Nelson and Bloomfield, '24) the magnitude of p at 0C. is almost identical with the value of 12,000 obtained in this work.

Mutarot at i o n
Mutarotation of the reaction products and the change of fructofuranose to fructopyranose occur during hydrolysis, but are not limiting factors determining hydrolysis rate as a function of temperature. The influence of mutarotation on the velocity constant may be determined by bringing mutarotation to completion before measuring the rotation. This was done by adding 10 cc. samples of the digest to 2 cc. 1.0 M Na,CO, at successive time intervals. The solution is allowed to stand until mutarotation is complete. With a solution of 16% sucrose and 4% Difco invertase ( p H 5.87, 22.C.) the velocity constant before mutarotation is 0.0137, which becomes 0.0268 after mutarotation. The complications introduced by mutarotation may be avoided by using very dilute invertase solutions. With a 16% sucrose solution containing 0.02% Difco invertase ( p H 5.82, 24C.) K = 0.00110. This value is not changed after mutarotation. When inversion is very slow mutarotation occurs relatively so rapidly that it has no

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IRWIN W. SIZER

influence upon the velocity constant. The temperature characteristic, however, is the same as that obtained for solutions of higher invertase concentrations. A p of 11,600 was procured, and this value was independent of temperature changes. The data presented in the tables and plotted in figures 3 and 3 indicate that a sevenfold change of inversion velocity brings about no alteration in p, although the relative influence of mutarotation upon the velocity constant changes with it. Further evidence of the subordinate role played by mutarotation is the following: The sugars produced from raffinose by the action of invertase mutarotate quite differently from those produced from sucrose, yet the temperature characteristics are identical for the two hydrolyses. Calculations from the data of Nelson and Beegle ('19) indicate that the mutarotation of glucose has a p=17,000, and for fructose is about 25,000, the latter value decreasing with temperature. Both these values are very different from the 11 of 12,000 f o r sucrose inversion with yeast invertase.

!Mechanism of the reactiovz The hydrolysis of sucrose catalyzed by invertase is best considered as a heterogenous reaction (White, '33 ; Nelson, ' 3 3 ) , since invertase has a high molecular weight (60,000 according to Moelwyn-Hughes, ' 3 3 ) , forms a colloidal solution, and is active even when adsorbed on the surface of carbon. The adsorption of the substrate or of the substrate and water onto the active surface of the enzyme could then be considered a necessary preliminary to hydrolysis. The lag period lasting for about 12% of the inversion might then be regarded as the time required for the reacting molecules to become absorbed on the catalytic surface of the invertase. The products of the reaction might also be adsorbed, and would be evidenced by a slowing down during the latter part of the hydrolysis, the inhibition phase. The onset of the inhibition phase of the inversion of sucrose by yeast invertase is posponed when a relatively higher substrate concentration is used. With 4% sucrose the inhibition phase begins after

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58% of the inversion is completed, with 16% sucrose after ?O%, and with 40% sucrose the inhibition phase begins after 83% of the reaction is finished. This suggests that some factor, other than the accumulation of reaction products, such a s enzyme destruction may also be important in impeding the reaction during the later stages of inversion. The velocity of inversion is a function of the substrate concentration. Nelson ('33) states that the velocity of inversion rises t o a maximum when the sucrose concentration is increased to 5 % ; it then decreases linearly with further increase in substrate concentration. The results of this work are in excellent agreement with this statement. Temperature has no effect upon the sucrose concentration at which the rate of hydrolysis reaches a maximum. This maximum at a particular substrate concentration is explained by assuming that the velocity of hydrolysis is conditioned not alone by the adsorption of sucrose on the enzyme surface, but by the adsorption of water a s well. Thus, at a particular sugar concentration the relative amounts of substrate and water adsorbed on the catalytic surface will be at an optimum, and the reaction will proceed a t its greatest velocity. Many have shown that inversion velocity is at a maximum at pH 4.7 (Haldane, '30), decreasing on either side of this point. Similar results were obtained in this investigation on yeast invertase. A change in temperature has no effect upon the pH a t which the maximum velocity of hydrolysis occurs. Thus the optimum substrate concentration and the optimum (H+) for the activity of yeast invertase are unaltered by changes in temperature. This would be expected if the mechanism of the reaction i s the same at different temperatures, as the constant temperature characteristic indicates.

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IRWIN W. SIZER

SUMMARY

1. The velocity of sugar inversion catalyzed by HC1 increases exponentially with temperature according to the Arrhenius equation. A p value of 26,000 was obtained, confirming the results of others. 2. Sucrose inversion catalyzed by yeast invertase follows the unimolecular equation for about two-thirds of the reaction. It deviates sharply from the theoretical curve at the beginning (the lag period) and toward the end of the reaction (the inhibition period). A I-I value of 12,000 is obtained when data on inversion velocity during the first two-thirds of the reaction are fitted to the Arrhenius equation. This value is constant over the temperature range from 4 to 45C. I t is practically the same for different samples of invertase, is unaffected by changes in sucrose or invertase concentration, is independent of pH changes in the range from 3.2 to 7.9 and is not altered by the presence of electrolytes. 3. Yeast invertase yields the same p value of 12,000 whether it is catalyzing the hydrolysis of raffinose or sucrose. This indicates that the p value is the energy of activation of the catalyst and is not determined by the nature of the substrate, but other substrates should be studied before making generalizations. 4 Invertase from malt is characteristically different from . that obtained from yeast. The lag period in the inversion is lacking, the temperature characteris'tic is 13,000, and the enzyme is inactivated above 35C.

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L I T E R A T U R E CITED AEBBENIUS, S. 1889 flber die Reaktionsgeschmindiglreit bei der Inversion von Rohrzucker durch Siuren. Z. Physik. Chem., Bd. 4, S. 226-248. AUDEN, H. A. AND E. R. DAWSON 1931 The hydrolysis of concentrated sugar solutions by invertase. Biochem. J., vol. 25, pp. 1909-1916. CROZIER, . J. 1924-1925 On biological oxidations as function of temperature. W J. Gen. Physiol., vol. 7, pp. 189-216. 1934 A Handbook of General Experimental CROEIER, W. J. AND H. HOAGLAND Psychology, chapt. 1. (Carl Murchison ed.) Clark University Press, Worcester. EULER, AND J. LAURIN1919-1920 Uber die Temperaturempfindlichkeit d e r H. Saccharase (Invertase). Z. Physiol. Chem., Bd. 108, S. 64-114. 1920 fiber den Temperaturkoeffizienten den Saccharasewirkung. Z. Physiol. Chem., Bd. 110, S . 55-92. EULER, AND B. UGGLAS 1910 Untersuchungen iiber die chemische ZusamH. mensetzung und Bildung der Enzyme. Z. Physiol. Chem., Bd. 65, S. 124-140. GLASSTONE,S. 1933 Recent Advances in Physical Chemistry. Blakiston, Philadelphia. HALDANE, B. S. 1930 Enzymes. Longmans, Green and Co., London. J. KERTDSZ, I. 1933 Wiirmetonungskonstante der Hefesaccharase in unterZ. kuhlten Liisungen. Physiol. Chem., Hd. 216, S. 229-232. MOELWYN-HUGITES, A. 1933 The kinetics of enzyme reactions. Ergebnisse E. d. Enzymforschung., Bd. 2, S. 1-22. NELSON,J. M. 1933 Enzymes from the standpoint of the chemistry of invertase. Chem. Rev., vol. 12, pp. 1-42. NELSON,J. M. AND F. M. BEEGLE1919 Mutarotation of glucose and fructose. J. Am. Chem. Soc., vol. 41, pp. 559-575. NELSON, J. M. AND G. BLOOMFIELD1924 Some characteristics of invertase action. J. Am. Chem. Soc., vol. 46, pp. 1025-1043. O'SULLIVAN,C. AND F. W. THOMPSON 1890 Invertase: A contribution t o t h e history of an enzyme or unorganized ferment. J. Chem. Roc., vol. 57, pp. 834-931. TAMMANN, . 1892 Die Reactionen der ungeformten Fermente. Physiol. Chem., G Bd. 16, S. 271-329. H. 1937 Enzyme Chemistry. J. Wiley and Sons, Inc., New York. TAUBER, WHITE, T. A. 1933 Invertase a s a heterogeneous reaction. J. Am. Chem. Soe., V O ~ .55, pp. 556-568.