EXCHANGE ' J9 PUlo Uniformity in Invcrtasc Action By DAVID INGERSOLL HITCHCOCK DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF Tim REQUIREMENTS FOR Tim DEGREE OF DOCTOR OF PHILOSOPHY IN THE FACULTY OF PURE SCIENCE, COLUMBIA UNIVERSITY Jfotu 1 ark -0.001975 2 -0.00002034 3 . (n) 100 p The applicability of this equation may be seen from the following figures . TABLE II. APPLICATION OP EMPIRICAL EQUATION. Inversion. P Time, /(calc.). Min. Time, /(obs.). n Min. x io. a 315 4.96 5 443 6.35 10.1 10 449 9.38 14.9 15 445 13.77 22.1 22 449 18.52 30.1 30 447 35.43 60.0 60 446 50.27 89.8 90 445 62.73 119.6 120 445 80.30 180.6 180 448 94.48 299.9 300 447 Mean, 446 A. d., 0.36%. a Meaning of n explained below. 11 Hudson, J. Am. Chem. Soc., 36, 1571 (1914). 12 IV. An Equation for Experiments with Different Amounts of Invertase. Nelson and Vosburgh 1 showed that, in their experiments in which the initial sucrose concentration was constant, the time required for a given percentage of sucrose to be inverted was inversely proportional to the amount of invertase used. In other words, letting t represent the time for 80% inversion, and y the invertase concentration, in experiments in which only the invertase concentration was varied they found the pro- duct ty to be constant. In another series of experiments with a different invertase preparation they called t the time for 40% inversion, and here again ty was constant. They did not, however, compare the times for different degrees of inversion in any one experiment, say t for 40% and t for 80% inversion, because they did not know the law governing the re- lationship between the time and the percentage of inversion, or, mathe- matically, the form of the function, t=i(p). It is obvious that one may plot the values for the amounts hydrolyzed in various times against the times, obtaining curves for the hydrolyses which are graphical representations of the function, t = i(p). This was done by Michaelis and Davidsohn 12 in such a way as to compare the form of the function in three experiments with invertase concentrations in the ratio 2:1: 0.4. They plotted the product of the enzyme concentration and the time, ty, against the change in rotation, which is proportional to the percentage inverted, p. They claimed that the points all fell on a smooth curve, and that therefore the form of the hydrolysis curve was independent of the amount of enzyme. However, only 3 experiments were given of which one was represented by only 2 points. Moreover, their whole curve did not appear to extend much beyond the first half of the inversion, and in addition several of their points did not fall on the curve, even on the small scale used in their printed article. Because of these deficiencies, and because the shape of the curve is a fundamental point in the present investigation, it seemed best to amplify their data by the use of the more extensive experiments of Nelson and Vosburgh. Accordingly the results of the latter were plotted in a similar way on a large scale. The curves were brought together at one point by using a different time scale for each experiment. When the remainder of each curve was plotted on this new scale, it was found that the curves for experi- ments with the same initial sucrose concentration did superimpose, falling on a single smooth curve. Thus the conclusion drawn by Michaelis and Davidsohn was more firmly established by the results of Expts. 6, 7, 8, 9, 10, 22 and 23 of Nelson and Vosburgh, each experiment including at least 6 samples and extending over 95% or more of the inversion. This means that the function, t = f (p), representing a single experiment, can be general- ized as nt = J? (p) for experiments with varying amounts of invertase^ 12 Michaelis and Davidsohn, Biochem. Z., 35, 386 (1911). 13 Here n is a constant in any one experiment, but varies in different experi- ments, being proportional to the amount of effective invertase. Moreover the form of the function nt F (p) is, within the limits of these experi- ments, independent of the amount of invertase or of the rate of the hy- drolysis. A more exact verification of this relationship was obtained by the use of Equation 11, which gives a definite form to the function, * = f (p), for one particular invertase concentration. In order to make this equation generally applicable to experiments with other invertase concentrations, the coefficient of the logarithmic term was placed equal to l/n and factored out, giving + 0.002642^-0. 000008860/> 2 -0.0000001034 3 ] . (12) i n 100 p If it is generally true that the times for any given percentage of inversion are inversely proportional to the amounts of invertase used, then Equa- tion 12 gives a definite form to the function, t = i (p), for any invertase concentration. Whether or not this is the case can be tested by substi- tuting in Equation 12 the experimental values for p and /, and calculating the values of n. If the latter are constant, the equation applies and the general law holds, and the values of n should be directly proportional to the amounts of active invertase present. To recapitulate, we have, if this is true, first an empirical relation be- tween time and percentage inverted which holds for experiments in which different amounts of invertase are used; and, second, we-have in the value of n a relative measure of the amount of the effective invertase. In order to decide whether or not Equation 12 applies to a given experi- ment, it is necessary to decide whether or not the values of n are constant. The values of n for the experiments of Table IA, from which the equation was derived, are given in the last column of Table II. The average de- viation from the mean, 0.36%, is an indication of the extent to which the equation fits these original experiments. To determine about what magni- tude of deviation from the mean might be due to experimental error in applying the equation to other experiments, the following calculations were made. From the agreement of duplicate experiments in Table I and subsequent experiments, the average error in determining any change in rotation was estimated as 0.02. To determine what error in n could be caused by such an error, the value 0.02 was added to all the changes in rotation of Table IA, and the values of n recalculated, with the results shown in Table III. Evidently the form of the relationship is such that errors are magnified in the values of n calculated from the data on the early part of the hy- drolysis. Since in all the experiments of the present work except those of Table I the first sample was taken at about 10% inversion, while the 14 TABLE III. EFFECT OF ASSUMED ERROR OP 0.02. P + error. t. 10* (n + error). 10 8 (n true Error in Dev. from values). 10*M. true mean. 3.26 5 458 443 15 12 6.47 10 457 449 8 11 9.50 15 450 445 5 4 13.89 22 453 449 4 7 18.64 30 450 447 3 4 35.55 60 448 446 2 2 50.39 90 . 446 445 1 62.85 120 446 445 1 80.42 180 450 448 2 4 94.60 300 450 447 3 4 Mean, 4. 4 = 0.99%. Mean,4.8 = 1.08%. other 7 samples were distributed about as before, it was decided that a fairer measure of the average error in n would be given by the mean of the last 8 of the above values. This gives an average deviation of 0.59% from the individual values of n, or of 0.70% from the mean value of n as the deviation caused by an error of 0.02 in the value of each change in rotation. Hence it may fairly be decided that any experiment giving an average deviation from the mean of 0.7% or less is fitted by the equation, and its curve has the same shape or the function, nt = T? (p), has the same form as in the case of the original experiments of Table IA. Since the results of Nelson and Vosburgh were available, including ex- periments in which the concentration of invertase was varied, it was thought that these results might well be used as a test of the general applicability of Equation 12. Accordingly Table IV was prepared by using those of their experiments in which the initial sucrose concentration was 10 g. per 100 cc. In the last three of these experiments the average deviation from the mean of the values of n is below the value 0.7%. As has been already pointed out, this deviation might be caused by experimental error, and accordingly the equation fits these three experiments satisfactorily. In Expts. 6, 8, and 9 the first sample was taken before the inversion was 10% complete. Now it has been already pointed out that in this part of the inversion a small experimental error may cau^se a large error in the value of n. Accordingly for these experiments the mean of the remaining values of n was calculated, omitting the first, and the average deviations were found to be 0.64%, 0.40%, and 0.10% for Expts. 6, 8 and 9, respectively. Therefore the equation really does fit 6 of the 7 experiments in Table IV, and it may be concluded with more certainty than before that the shape of the hydrolysis curve or the form of the function, n = F (p), isindepend- 15 TABLE IV. EFFECT OF VARYING AMOUNTS OF INVERTASE. (EXPERIMENTS OF NELSON AND Vos- BURGH.) Initial sucrose concentration, 10 g. per 100 cc. Hydrogen-ion concentration, 3.2 X 10 ~ 5 to 2.1 X 10 ~ 6 moles per liter. Temperature, 37. Invertase preparation A used in Expts. 6 to 10, Preparation B in Expts. 22 and 23. Inv. per Time. Expt. 100. /. Cc. Min. Amt. in- verted, P,%. xio. 66 14 8.32 422 30 17.92 433 70 39.85 437 120 61.90 437 185 80.59 439 320 94.97 431 Mean, 433 Av . dev., 1 . 04%. 8 4 20 8.31 295 45 18.69 301 105 41.17 302 175 62.11 301 265 80.09 303 450 94.85 305 Mean, 301 Av. dev., 0. 73%. 10 2 50 10.01 143 100 20.14 146 221 42.09 147 315 56.10 146 345 60.04 146 570 81.60 147 1365 98.06 (131) Mean, 146 Av. dev., .57%. 23 2 14 10.34 526 28 20.49 533 60 41.81 538 100 62.62 533 156 81.70 537 280 95.70 (517) Mean, 533 Inv. per Expt. 100. Cc. Time Amt. in- t. verted. Min. p, %. nX10. 7 5 22 11.08 360 40 20.15 366 90 43.21 372 138 60.98 372 215 80.30 375 373 95 '.04 370 Mean , 369 Av .dev., 1, 14%. 9 3 33 9.57 206 70 20.49 213 150 41.50 213 250 62.44 212 376 80.09 213 660 95.28 213 Mean , 212 Av. dev., 0. 80%. 22 1 30 10.91 259 60 21.50 261 122 41.21 260 200 61.49 260 305 79.65 260 600 96.08 (248) a Mean, 260 Av. dev., 0.17%. Av. dev., 0.60%. a These values are for points beyond the limit of p, 95%, for which the equation was derived, and hence were not used in taking the mean. ent of the invertase concentration, and that Equation 12 gives a definite form to this function, F(^). In view of the fact that the extreme variation in the invertase concen- tration in these experiments was from 6 cc. to 2 cc. or in the ratio 3:1, while the range covered by the rather unsatisfactory experiments of Michaelis and Davidsohn was 5 :1, it seemed best to try the effect of a wider variation 16 in invertase concentration on the shape of the curve. The highest con- centration used was selected so as to make the hydrolysis as rapid as possi- ble without causing error in the timing of samples, and the lowest concen- tration was such that the first and last samples could just conveniently be taken on the same day. In view of the difficulty encountered in a pre- vious investigation 13 in obtaining reproducible results with very dilute invertase solutions, it seemed unwise to attempt to study slower reactions than this. The results of the experiments with the extreme invertase concentrations used, in the ratio 12:1, are given in Table V. TABLE V. EXTREME CHANGES IN INVERTASE CONCENTRATION. Expts. B60 and B61. 6 cc. of Invertase 8 per 100 cc. Expt. B62. . 5 cc. of Invertase 8 per 100 cc. Time, t. Min. 5 Rotation, B60. B61. Degrees. 13.09 13.09 11.11 11.11 Amt. inverted, nXlO*. P, % 11.75 168 Time. t. Min. 60 Rotation, degrees. 13.04 11.11 Amt. inverted, nX10 B . P, % 11.45 136 10 9.24 9.25 22.85 167 120 9.28 22.31 136 15 7.50 7.51 33,18 166 180 7.58 32.40 135 21 5.57 5.58 44.63 166 252 5.68 43.68 135 28 3.59 3.59 56.38 165 336 3.76 55.07 134 37 1.49 1.49 68.84 166 444 1.70 67.30 133 52 -0.93 -0.94 83.26 168 624 -0.70 81.54 134 70 -2.43 -2.43 92.11 170 840 -2.26 90.80 134 1-7 days -3.76 -3.76 Mean, 167 11 days -3.81 Mean, 135 Av.dev., 0.75% Av. dev.,0.65% The values of n are sufficiently constant so that the equation may be said to hold for these concentrations. If the time for any given percentage of in version is inversely proportional to the concentration of invertase, the value of n divided by the number of cubic centimeters of invertase used per 100 cc. of solution should be a constant for any given invertase preparation. For Expts. Bl and B2 (Table IA) this value is 0.00292; for Vosburgh and Nelson's Expt. IB (Table VI), it is 0.00290; for B60 and B61, 0.00278; and for B62, 0.00270. These experiments were all made with Invertase 8. The difference be- tween the former two and the latter two values is due to slow deterioration of the invertase, even when kept in the ice-box, for a period of 8 months had elapsed between the two sets of experiments. The smaller difference between the latter two values can hardly be so explained, as the experi- ments were run on successive days, but must be taken to mean that for this range of concentrations the effective activity of the invertase is not strictly proportional to the actual concentration used. However, since the equation applies equally well in both cases, it may be stated as a fact 13 Nelson and Hitchcock, "The Activity of Adsorbed Invertase," J. Am. Chem. Soc., 43, 1956 (1921). 17 that over this range Q invertase concentrations (12: 1) the form of the function, wZ = F(), is the same and is expressed by Equation 12, while the value of n represents accurately the true activity of the invertase even better than its relative concentration. V. Effect of Temperature. Since the experiments of Nelson and Vosburgh were carried out at 37 while the present experiments were run at 25, it seemed that the effect of temperature differences in any two experiments might be constant for all stages of the reaction. Inasmuch as some experiments on the course of the hydrolysis at various temperatures had recently been made in this TABLE VI. EFFECT OF TEMPERATURE. (EXPERIMENTS OF VOSBURGH AND NELSON.) Initial sucrose concentration, 10 g. per 100 cc. Hydrogen-ion concentration, 4 . 4 X 10 ~ 5 to 4.0 X 10 ~ 5 moles per liter. Invertase preparation No. 8, 1 cc. per 100 cc. Expt. Temp. C Time, /. Min. Amt. inverted, P, % X10 5 . Expt. Temp. C. Time, t. Min. Amt. inverted, nXlO 11B 15 21 5.22 175 15B 20 16 5.04 222 63 14.54 166 38 11.87 223 110 24.39 163 65 19.82 222 162 34.72 162 120 34.90 220 250 50.45 161 155 43.68 219 350 65.04 161 190 51.40 217 441 75.07 161 254 63.92 216 586 85.88 162 320 74.18 217 Mean, 164 420 84.87 218 Av, , dev., 2.07%. Mean, 219 Av. dev., 0.96%. IB 25 15 6.17 290 5B 30 9 4.93 381 36 14.60 292 29 15.07 374 63 24.80 289 50 25.22 371 105 39.70 290 71 35.05 373 165 57.39 287 107 49.79 370 235 73.24 289 153 65.34 370 360 88.84 290 190 75.07 373 Mean , 290 246 84.93 374 Av . dev., 0. 34%. Mean, 373. Av. dev., 0.60%. 7B 35 8 24 42 60 90 120 155 195 5.46 15.76 26.59 36.91 52.11 64.63 76.02 84.93 482 473 467 467 465 464 467 471 Mean, 469 Av. dev., 0.91%. 18 laboratory by Vosburgh and Nelson, 14 it seemed inadvisable to repeat this work. Accordingly the effect of temperature on the shape of the hydrolysis curve was tested by applying Equation 12 to these experiments, with the results shown in Table VI. In all of these experiments the first sample was taken at a point con- siderably below 10% inversion. Therefore, in order to compare the average deviation with that which might be due to experimental error, the values obtained from the first sample should be omitted in taking the mean. If this is done the values for the average deviation are as follows: Kxpt. 11B, 0.68%; 15B, 0.91%; IB, 0.34%; 5B, 0.43%; and 7B, 0.56%. All of these except that for Expt. 15B are less than 0.7%, and accordingly Equation 12 fits these experiments fairly well. This establishes for the first time the fact that temperature differences, at least between 15 and 35, have no effect on the shape of the hydrolysis curve or the form of the function, nt = 'P (p). In other words, an increase in the temperature has the same quantitative effect as an increase in the amount of the inver- tase used. VI. Effect of Hydrogen-ion Concentration. In his classical study of the effect of hydrogen -ion concentration on in- vertase action, Sorensen 15 found that the velocity coefficient k calculated according to the unimolecular law in the form 1 axi k = In h h axz increased considerably as the reaction progressed in nearly neutral solu- tions (C H + = 10~ 6 to 10~ 7 ), increased less around the optimum (C H + = 10 ~ 4 to 10 ~ 5 ), remained constant in slightly more acid solutions (C H + = 1.2X10" 4 ), and decreased in still more acid solutions (C H + = 2.1X10~ 4 ). This means that the shape of the hydrolysis curve or the form of the func- tion nt = 3? (p) was not the same, in his experiments, for different hydrogen- ion concentrations. Michaelis and Davidsohn 12 have pointed out that this variation may be explained in part by destruction of the invertase in the more acid solutions at the rather high temperature, 52, at which Sorensen carried on his experiments. By using a lower temperature, 22.3, they obtained values of k calculated from the equation k - i log JL t a-x which increased in experiments at hydrogen-ion concentrations less than 3.0X10" 3 , where they remained constant. Nelson and Vosburgh, 1 on the other hand, found that at 37 the values of k increased in experiments 14 Vosburgh and Nelson, "The Temperature Coefficient of Invertase Action," (to be published later). 15 Sorensen, ^Biochem. Z., 21, 131-304 (1909); also Compt. rend. Lab. Carlsberg, 8, 1 (1909). 19 at the optimum hydrogen-ion concentration, 3.2X10" 6 , but increased more slowly or remained constant at 3. 2X10 ~ 6 . They noticed, however, that there were some changes in the hydrogen-ion concentration during the latter half of the inversion at about 3.2X10" 6 . The equation of the present work will not fit experiments in which the values of the unimolecular "" are constant or decrease, because it was derived for experiments for which the unimolecular '"k" increased. In order to test the effect of hydrogen-ion concentration on the shape of the curve the equation was applied to some recent experiments of Vosburgh and Nelson (to be published later) in which the hydrogen-ion concentra- tion was held constant at 10 ~ 6 moles per liter by means of citrate buffers and the improved procedure recommended by Vosburgh 16 was used. These results are given in Table VII. (EXPERIMENTS OF Vos- VII. EFFECT OF A DIFFERENT HYDROGEN-ION CONCENTRATION. BURGH AND NELSON.) Initial sucrose concentration, 10 g. per 100 cc. Hydrogen-ion concentration, 1 . 10 X 10 ~ 6 to 1 . 13 X 10 ~ 6 moles per liter. Invertase preparation No. 8, 1 cc. per 100 cc. Expt. Temp. C. Time, t. Min. Amt. inverted, P,%. nXlO'. Time Expt. Temp. t. C. Min. , Amt. inverted, P, %- nX10. 13B 15 23 4.99 153 2B 25 30 11,10 264 75 15.43 148 55 19.76 261 126 25.34 148 80 28.13 261 183 35.67 148 101 34.84 260 282 51.57 147 138 45.58 258 390 65. .46 146 217 64.87 258 490 75.43 146 350 84.69 261 642 85.52 146 Mean, 260 Mean, Av. dev., 1. 148 01%. Av. dev., .62%. 9B 35 10 5.88 415 26 15.01 415 44 24.75 413 64 35.10 414 98 50.56 412 135 64.45 411 171 74.96 413 225 85.52 416 Mean, 414 Av. dev., 0.34%. Except for the first value in Expt. 13B, the deviation of which may be due to a slight experimental error, as has been already pointed out, the constancy of n is very satisfactory. This means that at a hydrogen-ion concentration of 10 ~ 6 moles per liter the curve has the same shape or the 16 Vosburgh, "Some Errors in the Study of Invertase Action," /. Am. Chem. Soc., 43, 1693 (1921). 20 function nt = F(p) has the same form as at the optimum hydrogen-ion concentration. The differences found by Nelson and Vosburgh 1 at C H + 3!2X10~ 6 must be ascribed to changes in the hydrogen-ion concentration or in the amount of active invertase due to the use of hydrochloric acid without buffer. The nature of the buffer, however, does not seem to affect the shape of the curve, for the experiments of the present work were made with a 0.01 M buffer mixture of acetic acid and sodium acetate, while the experiments of Vosburgh and Nelson quoted in Tables VI and VII were made with a similar concentration of citric acid and sodium citrate. Very recently the range for which the equation holds has been extended to C H + 3.2X10" 7 by some experiments of Nelson and Bloom- field (not yet published). These results mean that within the limits given changes in hydrogen-ion concentration affect the activity of the invertase in just the same way as changes in temperature or in the amount of in- vertase used; either there is actually a change in the amount of the active substance present throughout the experiment or else the activity of the amount present is uniformly reduced or increased by the change and re- mains constant throughout the experiment. VII. A Criterion of Normal Invertase Action. Above, in Part II of this paper, experiments were given which showed that not all invertase preparations impart the same shape to the hydrolysis curve. Equation 12 was made to fit the experiments in Table IA, made with invertase preparations which were classified as normal. Accordingly it seemed probable that it would not fit the experiments in Table IB, and hence might be used as a means of distinguishing between normal and TABLE VIII. APPLICATION OP THE EQUATION AS A CRITERION OF NORMAL INVERTASE ACTION. Expts. B 12-15. Expts. B17 and B18. 1 .905 cc. of Invertase 3 per 100 cc. 10.45cc. of Invertase 6 per 100 cc. Time Min. Amt. inverted, P, % nX10. Time, t. Min. Rotation, B17. B18. Degrees. Amt. inverted, P.%. nXlO*. 13.15 13.15 5 3.20 450 15 11.47 11.46 10.03 476 10 6.35 449 30 9.88 9.88 19.41 470 15 9.44 448 50 7.90 7.90 31.16 466 22 13.65 445 70 6.10 6.10 41.84 461 30 18.46 446 90 4.52 4.49 51.34 457 60 34.96 440 115 2.75 61.72 454 90 49.38 436 140 1.35 1.32 70.15 451 120 61.48 433 180 -0.36 -0.40 80.30 448 180 78.81 431 240 -1.95 -1.96 89.67 449 300 93.59 426 300 -2.74 -2.74 94.30 443 Mean, 440 2 to 7 days -3.71 -3.71 Mean, 457 Av. dev., 1 .4%. Av. dev., 1.9%. , 21 abnormal invertase preparations. Equation 12, therefore, was applied to the results of Table IB, and also to experiments with two other invertase preparations, Nos. 6 and 7, with the results shown in Table VIII. Expts. B20 and B21. 3.60 cc. of Invertase 7 per 100 cc. Time, Rotation, Amt.- /. B20. B21. inverted, n X 10. Min. Degrees. p, %. 13.07 13.08 15 11.47 11.47 9.55 453 30 9.89 9.91 18.87 456 50 7.94 . 7.96 30.45 454 70 6.17 6.16 41.00 451 90 4.53 4.54 50.68 450 117 2.64 2.66 61.90 448 140 1.29 1.33 69.85 448 180 -0.49 -0.43 80.36 449 240 -2.06 -2.06 89.85 452 300 -2.85 -2.81 94.42 446 2 to 7 days -3.78 -3.78 Mean, 450 Av. dev., 0.56%. - It will be noticed in Expts. B 12-15, Table VIII, that for those points where the data of Table IB coincided with those of Table I A, or up to 20% of the inversion, the values of n are fairly constant, even for abnormal invertase. The abnormality, however, shows up later in the decreasing values of n, and is indicated by the larger values of the average deviation of a single value of n from the mean, which is well above 0.7% for the ex- periments with Invertase 3. Expts. B17 and B18 indicate that Invertase 6 is also an abnormal invertase preparation, since the values of n decrease and the average deviation is well above 0.7%. Invertase 7, on the other hand, is a normal invertase preparation, as is shown by Expts. B20 and B21, since the values of n exhibit satisfactory constancy. These experi- ments indicate that Equation 12 may be used as a criterion of normal invertase action. In order to decide whether invertase preparations are normal or abnormal, then, it is no longer necessary to use them at initially equivalent effective concentrations, but the experiments may be made with any concentration, at least within the limits of the experiments in Table V. If the average deviation of the values of n is under 0.7%, the invertase preparation may be classified as normal; if the values of n decrease and the average deviation is much over 0.7%, then the invertase preparation is abnormal. VIII. Attempts to Make the Abnormal Invertase Act Normally. There were no known differences in the method used in obtaining the normal and abnormal invertase preparations. However, it was deemed advisable to find out whether the abnormality could be due to some im- 22 purity which might be removed by further dialysis. Accordingly a sample of Invertase 6 was dialyzed for 3 days more in a collodion bag against running tap water. During the dialysis its volume was about doubled and its activity decreased by about x /2 on that account ; this was designated as Invertase 6B. To avoid this loss in activity, a sample of Invertase 3 was concentrated by evaporation in a collodion bag by fanning at room temperature 17 until it had lost about half its volume, and then dialyzed for 4 days, when it had regained about its original volume ; this was desig- nated as Invertase 3B. The results of experiments with these dialyzed preparations are given in Table IX. TABLE IX. EFFECT OF DIALYSIS ON ABNORMAL INVERTASE. Expt. B22. 16 cc. of Invertase 6B per 100 cc. Time, Amt. inverted, Expt. B24. Min. Rotation, degrees. 13.10 15 11.41 30 9.78 50 7.74 70 5.89 90 4.24 115 2.53 140 1.10 180 -0.58 240 -2.08 300 -2.83 nXlO*. Time, t. Min. Rotation, degrees. : SB per 100 cc. Amt. averted. nX10. 9.55 453 18.75 453 35.43 446 49.97 442 61.72 435 78.58 429 87.95 422 92.94 412 Mean, 437 ... 13.05 10.03 476 15 11.44 19.70 477 30 9.89 31.81 476 60 7.08 42.79 473 90 4.63 52.58 470 120 2.65 62.73 464 180 -0.19 71.22 462 240 -1.77 81.19 459 300 -2.61 90.09 456 4 days -3.80 94.54 449 Av. dev., 2.75%. Mean, 466 Av. dev., 1.76%. The decrease in the values of n and the large average deviations show that the invertase was still abnormal.. Since the abnormality could not be removed by purification by dialysis, it was thought that it might be due to the absence of some substance con- tained in the normal invertase. A sample of Invertase 8 was inactivated by boiling, and was proved to be totally inactive by the absence of any action on sugar solutions. Experiments were then conducted in which the solutions contained 10 cc. of this inactive invertase per 100 cc. in addi- tion to the abnormal invertase under investigation. The results are given in Table X. The figures in Table X indicate that the presence of boiled normal in- vertase caused preparation No. 3 to act normally, giving constant values of n, while it was practically without effect on preparation No. 6. This apparently means that there are different kinds of abnormality in different invertase preparations. 17 Kober, J. Am. Chem. Soc., 39, 944 (1917). 23 TABLE X. EFFECT OF BOILED NORMAL INVERTASE ON THE ACTION OF ABNORMAL INVERTASE. Expt. B25. 10 cc. of boiled Invertase 8 and 1.905cc. of Invertase 3 per 100 cc. Time, Amt. Rotation, inverted, X10. Min. degrees. P, % 13.17 . . 15 11.61 9.26 439 30 10.10 18.22 440 60 7.31 34.78 437 90 4.85 49.38 436 120 2.78 61.66 434 180 -0.19 79.29 437 240 -1.85 89.14 440 300 -2.68 94.07 437 3 days -3.68 Mean, 437 Av. dev., 0.34%. Expt. B26. 10 cc. of boiled Invertase 8 and 1 .943 cc. of Invertase 3 perlOO cc. Time, Min. Rotation, degrees. Amt. inverted, xio. 13.17 15 11. '59 9.38 445 30 10.06 18.46 446 60 7.24 35.19 443 90 4.76 49.91 441 120 2.67 62.31 441 180 -0.26 79.70 441 240 -1.91 89.50 446 300 -2.72 94.30 443 3days -3.71 Mean , 443 10.45 Expts. B36 and B38. cc. of Invertase 6 per 100 cc. Av. dev., 0.40%. Expts. B35 and B37. 10 cc. of boiled Invertase 8 and 10.45 cc. of Invertase 6 per 100 cc. Time, Min. Rotation, B36. B38. Degrees. Amt. inverted, P, % wX10. Time, Min. Rotation, Amt. B35. B37. inverted, nX10. Degrees. p, %. 13.14 13 ,13 . . . 13.25 13 .24 15 11.55 11 .57 9.38 445 15 11.67 11 .67 9.38 445 30 10.04 10 ,06 18.34 443 30 10.15 10 .16 18.34 443 60 7.27 7, ,29 34.78 437 60 7.36 7 .39 34.84 438 90 4.87 4.88 49.02 432 90 4.94 4 .98 49.20 434 120 2.84 2, .84 61.13 429 120 2.92 2 .94 61.25 430 180 -0.04 -0 06 78.28 426 180 -0.01 .04 78.52 428 240 -1.72 -1 .71 88.13 424 240 -1.67 -1 .64 88.43 429 300 -2.61 -2 59 93.41 422 300 -2.53 -2 .50 93.53 425 3-6 days, ,-3.72 -3 .72 Av. Mean , dev., 1 , 433 .67%. 2-5 das. -3.60 o .61 Av. Mean dev. 1. , 434 38%. In order to show that preparation No. 3 had not become normal simply on standing, but that the normal course of the reaction was really pro- duced by the presence of the boiled invertase, other experiments were run with Invertase 3 alone, and were found to give decreasing values of n, as before. These values, however, were somewhat smaller than those obtained in Expts. B12 to B15, indicating that this preparation had appreciably lost activity on being kept in the ice-box for less than 5 months. Further experiments were made with the abnormal invertase prepa- rations Nos. 3 and 6 in the presence of different concentrations of sodium chloride. The results are given in Table XI. The figures in Table XI indicate that increasing concentrations of sodium chloride exert an increasing retarding effect on the action of the 24 TABLE XI. EFFECT OF SODIUM CHLORIDE ON ABNORMAL INVERTASE. Expt. B44. Expt. B43. 1 .905 cc. of Invertase 3 per 100 cc. in .02 M NaCl. 1 .905 cc. of Invertase 3 per 100 cc. in 0.05 M NaCl. Time, /. Min. 17 Rotation, degrees. 13.05 11.34 Amt. inverted, wXlO 8 . P, % Time, /. Min. 15 Rotation, degrees. 13.05 11.54 Amt. inverted, X10*. P, % 8.96 424 10.15 425 30 10.08 17.63 425 30 10.12 17.39 419 60 7.38 33.65 422 60 7.44 33.29 417 90 4.99 47.83 420 90 5.08 47.30 414 120 2.99 59.70 416 120 3.03 59.47 414 180 0.00 77.45 417 180 0.05 77.15 414 240 -1.73 87.72 419 240 -1.69 1 87.48 415 300 -2.64 93.14 416 300 -2.61 92.94 412 2-3 days -3.80 Mean, 420 2-4 days -3.82 Mean , 416 Av. dev., . 71%. Av. dev., 0. 70%. Expt. B42. 1 .905 cc. of Invertase 3 per 100 cc. in . 1 M NaCl. Expt. B48. 10 .45 cc. of Invertase 6 per 100 cc. in 0.1 MNaCl. Time, t. Min. Rotation, degrees. Amt. inverted, P,%. HXIO'. Time, t. Min. Rotation, . degrees. Amt. inverted P, %. WX10S. 13.05 'o . . . 13.13 15 11.58 8.72 413 15 11.61 9.02 427 30 10.16 17.15 413 30 10.17 17.57 423 60 7.50 32.94 410 60 7.52 33.29 417 90 5.14 46.94 410 90 5.19 47.12 412 120 3.13 58.87 408 - 123 3.02 60.00 408 180 0.14 76.62 408 165 0.86 72.82 407 240 -1.66 87.30 413 225 -1.17 84.87 408 300 -2.59 92.82 410 300 -2.47 92.58 405 3 days -3.84 Mean, 411 3-6 days -3.75 Mean , 414 Av. dev., 0.46%. Av. dev., 1.67%. invertase. This was not observed in the work of Fales and Nelson 18 at the optimum hydrogen-ion concentration, but this may be due to the fact that they worked with a very much smaller sugar concentration, 0.5 g. per 100 cc. This retardation, however, seems to have more effect at the beginning of the hydrolysis than at the end in the case of Invertase 3, for in Expt. B42, with 0.1 M sodium chloride, the values of n were constant and the action must be classed as that of normal invertase. Invertase 6, however, was not made normal by 0.1 M sodium chloride, for in Expt. B48 the values of n decreased as much as ever. An experiment with 0.5 M sodium chloride and Invertase 6 gave values which decreased some- what less, but still were not constant enough for the action to be regarded 18 Fales and Nelson, J. Am. Chem. Soc., 37, 2769 (1915). 25 as normal. Unfortunately the supply of Invertase 6 became too low for further experiments to be carried out with it. Further experiments were made with Invertase 3 to determine the effect of invertase concentration on the abnormal action. The results are given in Table XII. TABLE XII. ABNORMAL INVERTASE AT DIFFERENT CONCENTRATIONS. Expts. B58 and B59. .5 cc. of Invertase 3 per 100 cc. Expts. B54 and B55. 3 cc. of Invertase 3 per 100 cc. Time, /. Min. Rotation, Amt. B58. B59. inverted, nXlQ 6 . Degrees. p, %. Time, Min. Rotation, B54. B55. Degrees. Amt. inverted, P, %. xio. 13 .05 13.05 . . . ' 13.06 13.06 60 11 .53 11.51 9 .08 108 10 11.46 11.48 9.44 671 120 10 .06 10.05 17 .80 107 20 9.96 9.98 18.34 664 195 8 .35 8.33 27 .95 106 30 8.52 8.55 26.88 662 270 6 .72 6.77 37 .45 106 45 6.50 6.53 38.87 660 360 5 .06 5.06 47 .42 104 70 3.62 3.67 55.91 654 450 3 .50 3.54 56 .56 103 100 0.98 71.69 654 540 2 .22 2.20 64 .33 102 120 -0.29 -0.28 79.23 654 1101 -1. 96 .... 89 .08 96 150 -1.62 -1.62 87.12 656 7 to 12 Mean, 104 3 to 8 Mean, 659 days -3 .79 -3.79 Av. dev., 2.7% days -3.79 -3.79 Av. dev., .74%. Time, f t B56. Min. 13.12 6 11.21 12 9.40 18 7.72 26 5.66 35 3.62 48 1.25 65 -0.88 85 -2.26 4 to 5 days -3.75 Expts. B56 and B57. 6 cc. of Invertase 3 per 100 cc. Rotation, Degrees. B57. Amt. inverted, MX10. 13.12 11.23 11.28 134 9.43 22.02 134 7.72 32.05 133 5.66 44.27 133 3.62 56.38 132 1.30 70.33 132 -0.84 82.97 134 91 .28 135 -3.74 Mean, 133 Av. dev., 0.67%. These results show that in the case of Invertase 3 the abnormality decreases with increasing amount of invertase or increases with decreasing amount of invertase or increasing time of reaction. It is not possible to explain these changes in the abnormality of Inver- tase 3 at the present time. IX. Attempts to Make Normal Invertase Become Abnormal. Since the presence of sodium chloride had seemed to some extent to favor the normal course of invertase action, it seemed worth while to find out whether further dialysis, by removing any last traces of salt, could 26 make a specimen of normal invertase act abnormally. Accordingly a sample of Invertase 8 was dialyzed for a week in a collodion bag against 8 changes of distilled water. This was designated as Invertase 8A, and when tested was found to be still normal, as is shown by the results in Table XIII, Expt. B46. Another sample of Invertase 8 was partially inactivated by heating for 1 hour on a water-bath at 50, and then for l /z hour more at about 57. This reduced its activity by about one-half. This invertase, No. 8E, was also found to be still normal, as is shown by Expts. B50 and B51, Table XII. ' A further attempt to render Invertase 8 abnormal was made by ex- posing some of it for 2 hours, in a quartz flask, to the ultraviolet and other radiation given by a mercury arc lamp. The result, Invertase 8F, had about one-half the activity of Invertase 8, but was also found to be still normal, as is shown by Expts. B52 and B53, Table XIII. TABUS XIII. ACTION OF NORMAL INVERTASE AFTER FURTHER DIALYSIS, HEATING, AND EXPOSURE TO THE MERCURY ARC. Expt. B46. Invertase 8A, dialysed, 5 cc. per 100 cc. Time, Amt. t. Rotation, inverted, nX10 e . Min. Degrees. p, %. 13.06 Expts. B50 and B51. Invertase 8E, heated, 5 cc. per 100 cc. Time, Rotation, Amt. /. B50. B51. inverted nX10. Min. Degrees. p, %. 13.10 13.10 15 11.27 10.62 505 15 10.94 10.94 12.82 612 30 9.56 20.77 504 30 8.88 8.89 25.04 614 60 6.44 39.29 501 50 6.37 6.38 39.94 612 90 3.78 55.07 499 70 4.18 4.19 52.94 610 120 1.66 67.66 497 90 2.30 2.35 63.98 610 180 -1.14 84.27 501 110 0.79 0.84 72.94 612 230 -2.34 91.39 502 140 -0.87 -0.85 82.85 618 270 -2.87 94.54 499 180 -2.20 -2.18 90.74 624 3 days -3.79 Mean, 501 2-5 days -3.78 -3.78 Mean, 614 Av. dev., 0.40%. Av. dev., 0.57%. Expts. B52 and B53. Invertase 8F, exposed to mercury arc, 5 cc. per 100 cc. Time, Rotation, Amt. /. B52. B53. inverted, nXlO*. Min. Degrees. P, % 13.09 13.09 15 10.33 10.35 16.32 785 25 8.61 8.63 26.53 783 35 6.99 7.04 36.08 781 45 5.51 5.53 44.92 779 65 2.93 2.99 60.12 775 85 0.94 0.97 "72.05 776 105 -0.54 -0.53 80.89 781 140 -2.12 -2.12 90.27 787 1 to 3 days 3.77 3 80 Mean, 781 Av. dev., 0.38%. 27 Since the values of n in Table XIII are constant in each experiment, having an average deviation from the mean in each case of less than 0.7%, the results show that the invertase was still acting normally. Hence it may be concluded that it is not possible by any of these three methods of treatment to render a normal invertase preparation abnormal. X. Experimental Details. Preparation of materials. The invertase used was all obtained from yeast by the method of Nelson and Born, 19 with slight modifications as described below. Preparations 6 and 7 had been made by previous workers in this laboratory and had been kept for several years in solution, saturated with toluene, in an ice-box. Preparations 1, 2 and 3 were made from yeast which had been permitted to autolyze for about a month and then filtered, and the filtrate had been treated with toluene and kept in stoppered bottles at room temperature for three years or more. During this time more solid matter had separated, and this was filtered off and the filtrate treated according to the method described by Nelson and Born 19 with the following modifications. Only one precipitation with alcohol was used and the kaolin treatment was omitted. After treatment with lead acetate and potassium oxalate, the filtrate was dialyzed for from 4 to 6 days in collodion bags against running tap water. The solutions become colorless and nearly clear during the dialysis. The dialyzed solutions were not precipitated again, but were preserved with toluene and kept in the ice-box until needed for the experiments. Preparation No. 8 was prepared by the same method from a new lot of 100 pounds of pressed yeast. 2Q The preparation of Invertase No. 2 was carried out by Nelson and Simons, 21 who modified the treatment further by nearly neutralizing the solution with ammonia before the alcohol precipitation. No differences in the method of preparation are known which might account for the abnor- mality of invertase preparations Nos. 3 and 6. Two lots of sucrose were used. In each case the starting point was the best com- mercial sugar, which was dissolved in distilled water and clarified with charcoal. The first lot was precipitated by alcohol by the method of Cohen and Commelin. 22 Its rotation was found to agree within 0.1% with that calculated from the formulas of Landolt and Schonrock. 23 The second lot was recrystallized from water by a procedure similar to that of Bates and Jackson. 24 Its rotation agreed with the calculated value within 0.04%. Other chemicals were c. p. grades, used without further purification. 19 Nelson and Born, J. Am. Chem. Soc., 36, 393 (1914). 20 Kindly furnished by the Jacob Ruppert Brewery of New York City. 21 Simons, Dissertation, Columbia University, 1921; Nelson and Simons, J. Am. Chem. Soc., (to be published later). 22 Cohen and Commelin, Z. physik. Chem., 64, 29 (1908). 23 Browne, "A Handbook of Sugar Analysis," John Wiley and Sons, New York, 1912, pp. 177-8. 24 Bates and Jackson, Bur. of Standards Sci. Papers, No. 268, 75 (1916). 28 Apparatus. Constant temperature was obtained by the use of an electrically controlled water-bath which remained at 25 0.01. The progress of the inversion was followed by means of a Schmidt and Haensch polarimeter reading to 0.01. The tubes used were 200 mm. long, and were proved to be of the same length by observing the rotation of the same 10% sugar solution in each tube. The temperature of the tubes was kept constant by the. thermostat described by Nelson and Beegle, 25 which maintained a temperature of 25 0.05. Monochromatic light of wave length 546.1 /*// was obtained from a mercury vapor arc by purification through two Wratten filters, one a No. 77, and the other a No. 77 which had been re-cemented with a green film in place of the yellow one. Thanks are due to Dr. C. B. K. Mees of the Eastman Kodak Company for preparing these filters. This light made it possible to use the polariscope with a half -shadow angle of 0.5. Nonsol bottles were used to contain the solutions undergoing hydrolysis. All volumetric apparatus used in making up solutions was calibrated. Control of the Hydrogen-ion Concentration. The desired hydrogen-ion concentration was obtained by the use of a buffer mixture of 0.1 M acetic acid and 0.1 M sodium acetate in the proportions given by Michaelis. 26 One hundred cc. of the final solution always contained 10 cc. of this buffer, making the total concentration 0.01 M. This concentration was low enough so that any salt effect on the invertase action was negligible, espec- ially at the optimum hydrogen-ion concentration. 18 In the experiments of Nelson and Vosburgh 1 the desired hydrogen-ion concentration was obtained by the use of diluted hydrochloric acid. In the experiments of Vosburgh and Nelson 14 a buffer of citric acid and secondary sodium citrate was used at a total citrate concentration of 0.01 M. The hydrogen-ion concentration was measured during or after each inversion by the colorimetric method of Sorensen, 15 using a-naphthyl- amino-azo-^-benzene sulfonic acid as indicator with citrate standards. The latter were standardized electrometrically with the hydrogen electrode and the saturated potassium chloride calomel cell 27 using a salt bridge of saturated potassium chloride solution. The hydrogen-ion concentrations were based on 0.1000 M hydrochloric acid as a standard, its ionization 28 being taken as 92.04% at 25, the temperature at which the present deter- minations were made. Procedure. In general the procedure followed was that recommended by Vosburgh. 16 Duplicate experiments were run on different days with freshly prepared sugar solutions. A solution was made up containing 25 Nelson and Beegle, /. Am. Chem. Soc., 41, 559 (1919). 26 Michaelis, "Die Wasserstoffionenkonzentration," Springer, Berlin, 1914, p. 184. 27 Kales and Mudge, /. Am. Chem. Soc., 42, 2434 (1920). 28 Fales and Vosburgh, ibid., 40, 1295 (1918). 29 sucrose and buffer in such concentrations that when a certain volume of this had been measured out it would be possible to add from a pipet a round number of cubic centimeters of invertase to start the reaction. For ex- ample, in Bxpt. Bl 32.680 g. of sucrose and 32.68 cc. of buffer were diluted to 500 cc. at 25 . Of this solution 321.80 cc. was pipetted into a Nonsol bottle, and 5 cc. of invertase was added to start the reaction. This produced the initial concentrations given in Table I A. The solutions were stirred by a current of filtered air while being mixed, and. samples were taken by pipets delivering in 10 seconds or less. 21 The time of mixing or of sampling was taken as the mean time of delivery of the pipet used. The reaction was stopped and mutarotation hastened by the use of sodium carbonate as recommended by Hudson, 29 a 25cc. sample being added to 5 cc. of 0.1 M sodium carbonate solution. The initial rotation of each solution was determined by preparing samples of identical composition in which the sodium carbonate was added to the sugar before the addition of the invertase, thus rendering the invertase entirely inactive. The rotation of each solution was determined by taking the mean of at least four concordant readings, the tube being rotated slightly after each reading to ensure the detection of any strain in the cover glasses. 30 The zero point of the polariscope was similarly determined by the use of a tube filled with distilled water. The final rotations were obtained by taking samples 2 days or more after the start of the reaction. Samples taken on the second and third days usually had the same rotation. In calculating the per- centage inverted, the total change in rotation was always taken as 16.85, since this value was obtained in all the experiments of Vosburgh and Nelson 14 as well as in the majority of the present experiments. Since in several experiments the total change in rotation appeared to be a few hundredths of a degree more than 16.85, it was thought ad- visable to test the effect of such differences on the values of n as obtained by the use of Equation 12. A sample calculation was made for Expt. B46, Table XIII, with the following results. Using 16.85, Using 16.89, nX10. nXlO*. 505 501 504 498 504 502 503 497 501 499 500 492 499 Mean, 501 497 Mean, 498 497 Av.dev.,0.40% 495 Av. dev.,0.60%. These results show that an error of 0.04 in determining the total change in rotation could not have caused sufficient error in the values of n to make a normal invertase preparation appear abnormal. Since this was the 29 Hudson, /. Am. Chem. Soc., 30, 1564 (1908). 80 Browne, Ref. 23, p. 156. 30 extreme deviation noticed from the value 16.85, the procedure adopted of taking the total change as 16.85 in all calculations is quite justified. A calculation of the possible error in determining the rotation of any sample which might be due to errors in the various measurements of weight and volume involved in this procedure has been made by Messrs. G. Bloom- field and F. Hollander of this laboratory. Using estimates of these errors based on the present authors' calibrations, this calculation gave a maxi- mum error of about 0.01 in the determination of the rotation of a sample. Since the duplicate experiments did not always agree so well as this, a fairer estimate of the precision of the measurements may be obtained from the agreement of the duplicates themselves. This would put the average difference between duplicate measurements of a change in rotation at about 0.02. The effect of such an error on the values of n obtained by the use of Equation 12 has already been considered. Summary. 1. It has been shown that not all preparations of yeast invertase are alike in their action, but that some are abnormal in allowing the hydrolysis of cane sugar to slow up more than others after the first 20% of the inver- sion. 2. An empirical equation is given which fits the hydrolysis of cane sugar by normal invertase over an extreme range of invertase concentration of 12 :1. By this means it has been shown that the hydrolysis-time curves for normal invertase are of the same shape for these different invertase concentrations and can be made to superimpose if the time scale be multi- plied by the proper constant. 3. By the same method it has been shown that the hydrolysis curve with normal invertase has the same shape at temperatures varying from 15 to 35, and at hydrogen-ion concentrations from 4.0 X 10~ 5 to 3.2 X 10~ 7 . 4. It was found that one abnormal invertase preparation could be ren- dered normal by the presence of .boiled normal invertase or 0.1 M sodium chloride, while another was not affected by either. The former preparation also worked normally at a very high concentration. 5. It was found impossible to render a normal invertase preparation abnormal by further dialysis or partial inactivation by heating or ultra- violet light. VITA. David Ingersoll Hitchcock was born in Detroit, Michigan, on June 26, 1893. In 1911 he was graduated from the Detroit Central High School. In 1915 he was graduated from Dartmouth College with the degree of Bachelor of Arts. From 1915 to 1917 he was Instructor in Chemistry at Dartmouth College. He was a graduate student in Chemistry at Columbia University during the summers of 1915, 1916, and 1917. In August, 1917, he enlisted in the 101st Machine Gun Battalion of the 26th Division, United States Army. In June, 1918, he was transferred to the Gas Service, later the Chemical Warfare Service, and was assigned to the chemical laboratory "at Hanlon Field, Chaumont, France. In January, 1919,' he was discharged from the Army. Since February, 1919, he has continued his studies at Columbia University, where he received the degree of Master of Arts in 1919. He has been laboratory assistant in various courses in the Department of Chemistry. Since February, 1920, he has been Harri- man Research Assistant in the laboratory of Professor J. M. Nelson. He is co-author with Professor Nelson of a paper on "The Activity of Ad- sorbed Invertase," which has been accepted for publication in the Journal of the American Chemical Society. For the year 1921-1922 he has been appointed a Fellow of the Rockefeller Institute for Medical Research, New York City. THIS BOOK IS DUE ON THE LAST DATE STAMPED BELOW AN INITIAL FINE OF 25 CENTS WILL BE ASSESSED FOR FAILURE TO RETURN THIS BOOK ON THE DATE DUE. THE PENALTY WILL INCREASE TO SO CENTS ON THE FOURTH DAY AND TO $t.OO ON THE SEVENTH DAY OVERDUE. NOV 2 1939 NQV # ,, V . ***, J, ?0,_ LD 21-100m-7,'39(402s) 478726 /J( UNIVERSITY OF CALIFORNIA LIBRARY