A New Method for Determining Invertase Activity W. R. JOHNSTON,SUTTON REDFERN, AND G. E. MILLER,The Fleischmann Laboratories, New York, N. Y.
T
HERE are several meth-
now in use for ing invertase activity (4, 7,9,11,16,IS). Thesemethods have as a basis the determination of either the unimolecular Ods
constantkortimevaluesrequired for a given percentage hydrolysis by a definite amount of invertase preparation, In addition, Nelson and Hitchcock (7) have pro-
A new method for the determination of yeast invertase has as a basis the determination of the number of enzyme units, termed 66invertons,9y per gram Of preparation. The enzyme Units are based upon the initial rate of inversion of the sucrose substrate. They are quickly and accurately evaluated, and are of practical Significance with respect to the actual performance of the enzyme. The method has been applied to three digerent commercial preparations with excellent results.
posed a n empirical c o n s t a n t which is proportional to enzyme concentration over a wide range of concentration. The methods utilizing the unimolecular k are not valid, as has been demonstrated repeatedly by several investigators employing time values are sufficiently (1, 6,8>*The accurate, but in general are time-consuming with respect to experimental measurements and calculations. The empirical constantof ~~l~~~and Hitchcock (7) is not easily evaluated, and while it is proportional to enzyme concentration it is not easilv exmessed in terms of actual enzvme Derformance. In the" authors' study of invertase they have-developed a method of activity measurement which is characterized by extreme simplicity of measurement and calculation. The method accurately evaluates enzyme activity in terms of the rate at which the invertase hydrolyzes the sucrose, thus giving an activity measure which is directly related to enzyme performance. It has been established by several workers (6, 8) that the initial rate of hydrolysis of sucrose by yeast invertase is directly proportional to the concentration of the invertase. The authors have elaborated on this work by studying the rate of hydrolysis very near the start of the reaction and have shown conclusively that the rate of hydrolysis a t zero t i m e that is, a t the very beginning of the inversion-is directly proportional to enzyme concentration over a wide range. Using this as a basis they have defined a rational enzyme unit termed the "inverton." The "inverton" is that amount of yeast invertase which will hydrolyze sucrose at the rate of 5 mg. per minute at zero time, under the specified experimental conditions. Since initial rates are proportional to invertase concentrations, we may use the inverton unit as a measure of the concentration of enzyme in a given preparation. The number of invertons per gram of preparation give a rational and logical measure of the strength of the preparation. This method of treatment has been previously applied to the study of alpha-amylase by Johnston and Jozsa, whose results will soon be published.
water. A commercial e n z y m e preparation preserved in g1 cerol was used. This was di1utedd.k use
~ y d ~ ~ ~ ~ l ~~ g~ $ water. The dilute enz me solu-
The initial reaction mixture corresponds to a 5 per cent sucrose solution, which is the optimum concentration for invertase act,ivity. The data in Table I were used in plotting the rate curves for the preparation used as a standard.
TIME Min.
1
2 3 4 6 8 10
RATE OF INVERSION BY
Invert,ase Conon. 2.5 mg./cc.
Mg. 31 66 101 137 208 279 350
Mg. 36 71 101
137 208 279 360
STANDARD INVERT.4SE
SUCROEE INVERTED Invertase Concn. Invertase Concn. 5 mg./oc. 10 mg./cc. Me. Mu. Mg. 67 67 141 139 138 281 210 206 282 so5 417 278 410 744 517 540 962 666 624 1197
Plotted on a large scale, the rate curves indicate that over the range tested the initial rate of action of the invertase is directly proportional to its concentration. This is illustrated in Figure 1. The results of Table I1 were obtained from the large-scale curves. TABLE11. INITIAL INVERSION RATESOF STANDARD INVERTASE HATEOF INVERSION OB CONCENTRATION OF INVERTASII PREPARATION SUCROSE AT ZEROTIME Mg./ml. Mg./min. 2.6 36 5.0 70 10.0 143
These results enable us to choose a rational enzyme unit which may be applied over the experimental range. In order to obtain a value approximating 100 enzyme units or invertons per gram for the standard commercial sample, the authors chose the inverton as equal to a rate of inversion of sucrose of 5 mg. per minute at zero time a t 25" C., under the conditions specified. This gives 112 enzyme units per gram of the standard invertase. To determine the inverton concentration of a given sample without resorting to rate measurements, it is necessary to
EXPERIMENTAL In order to establish the validity of the inverton, it is necessary to carry out careful measurements of initial rates of inversion. The measurements were made on a 5 per cent sucrose solution which was buffered to a pH of 4.6 by a Walpole acetate buffer (2). The sucrose solution was prepared by dissolving 100 grams of sucrose in distilled water, adding 50 ml. of Walpole acetate buffer of pH 4.6, and making up t o 1 liter at 25" C. with distilled a2
t
~
~ ~ $ ~ ~ ~ ~ ~
trate per ml. of solution. In carrying out a rate measurement 25 ml. of diluted sample were pipetted at 20' C.into a 200-m1. flask. The sample was placed in a thermostat at 25" C. and after coming to temperature, 25 ml. of a IOper cent sucrose solution were added at 25" c.,noting the time. The solution was added from a fast pipet a n d t h e mixture thoroughly shaken. The mixture was incubated at 25" C. for intervalsof 112, 3J4,6,8,and 10 minutes. The reaction was stopped and mutarotation accelerated by the addition of 0.5 ml. of 15 N ammonia. After standing for several minutes (longer than 5 minutes, less than 2 hours) the solutions were polarized in a 4-dm. tube, using a Fric saccharimeter with a Ventzke scale. The initial or blank polarization was determined in each case by polarizing a mixture of 25 ml. of sample, 0.5 ml. of ammonia, and 25 ml. of solution, mixed in the order named.
TABLE I.
$
March 15, 1935
ANALYTICAL EDITION
83
establish a relation beFor the range from 1025 to 2000 mg. a logarithmic equatween some easily obtain- tion was derived : able quantityand the numlog I = 0.0004289S 0.4490 ber of invertons present. S and I have their previous significance. The amount of sucrose inIn using these equations, one first calculates the number of verted after 0.5 hour a t 25" was chosen as the easily milligrams of sucrose inverted and then chooses the proper obtainable quantity. Di- equation to fit the sucrose value. The inverton value calculutions of the standard lated represents the number of invertons per 25 ml. of diluted invertase were made and sample. It must be calculated to a basis of 1 gram of original solutions of known inver- sample; so the dilution factor must be applied to the inverton t o n concentration were value obtained from the equation. For example, if 10 inverused a s o u t l i n e d pre- tons are calculated per 25 ml. of diluted sample and the diviously, except that the re- luted sample contains 125 mg. of original preparation per 25 action was stopped after ml., there are 80 invertons per gram of original sample. 30 minutes in each case TIME IN MINUTES TABLEIV. RATEOF INVERSION BY INVERTABE No. 1 instead of after variable (2.5 mg. of invertase preortration per ml. used in experiments A, B, and C; FIGURE 1. RATECURVES FOR 5 mp. per ml. in D and E) time intervals. STANDARD INVERTASE SUCROBE INVERTED A , 10.0 mg. per ml. Dilutions were made so A B C Av. U E Av. B, 5.0 mg. per ml. that the concentration Min. MO. MO. MO. MO. M O . M ~ . M ~ . C, 2.5 mg. per ml. 1 41.1 41.1 36.0 39.4 72.0 82.1 77.1 varied from 1.4 to 33.6 in2 71.8 71.8 72.0 71.9 144 144 144 vertons per 25 ml. of solution, corresponding to a variation of 3 108 113 108 110 211 221 216 4 139 144 144 142 278 277 278 0.5 to 12 mg. of standard invertase per 25 ml. 6 200 216 206 207 412 411 412 273 277 282 277 535 539 537 Calibrated glassware (Bureau of Standards) and distilled 8 344 349 343 345 659 657 658 water were used in all these J, dilutions in order to inIn order to establish the sure accuracy. Vosburgh ,~ method of measurement (10) found that errors were ,~ it was necessary to study introduced upon dilution the rate curves of other 26 with water c o n t a i n i n g commercial invertases and small amounts of electro11 investigate the constancy lytes. If an accuracy of Is of the calculated inverton 1 per cent is to be realized, 2o value on dilution of the distilled water must be ,e enzyme p r e p a r a t i o n . used. In carrying out the Three commercial invertdeterminations a standard S,+ ases were studied. It was 25-ml. pipet having an outfound that the activity of flow time of 35 seconds a t e,o all three invertases could 25" C. was used. Time be accurately determined was counted from the moby applying the inverton gc ment of introduction of %+ method. The results obthe sucrose. The mixture tained with commercialinwas shaken vigorously duro vertase No. 1 are given in MG. SUCROSE INVEATfD RFTER 30 MINUTES ing the addition. Table IV. The values of Table I11 FIGURE 2. INVERTON-SUCROSE CURVE The data for invertase were used in plotting the No. 1 were plotted in inverton-sucrose curve shown in Figure 2. Figure 3. The initial slopes of these rate curves showed the same property as those of the standard curves. The invertase concentration was diTABLE111. SUCROSE INVERSION AS FUNCTION O F INVERTON CONCENTRATION rectly proportional to the rate of inversion at zero SUCROSE TNVERT~DAFTER~~ INVERTONB MINUTEB 6W time. In order to check PER 2s ML. A B the behavior of the prepaMo. Me. SW ration on dilution the data 1.4 209 2.1 3i5 321 given inTable V(invertase 2.8 422 423 4.2 604 606 5 +m No. 1) were obtained. 5 6 782 784 8.4 1107 1115 k When plotted, the re11.2 1392 1395 g 100 sults in Table V gave the 16.8 1813 1818 z 22.4 2072 .. W straight line of Figure 4, 33.6 2331 .. z m showing clearly the linear w relation between concentration and number of inIt was found that the curve could be best fitted by two = equations. A least-squares solution for the portion of the vertons. curve up to 1025 mg. gave the following equation: Another commercial inTIME IN MINUTES FIGURE 3. RATE CURVES FOR vertase, No' 2, was purlog I = 1.0667 log S - 2.3368 chased in the open market INVERTASE No. 1 and checked exactly as inI = number of invertons per 25 ml. of sample A , 5.0 mg. per ml. X = mg. of sucrose inverted after 0.5 hour reaction1at 25' C. vertase No. 1 (Table VI B , 2.5 mg per ml.
+
.,6
$2
0
700
0
CD 100
INDUSTRIAL AND ENGINEERING CHEMISTRY
84 II
and Figure 5 ) . The same concordance I6 with the standard inv e r t a s e was ob,t served. The dilution check was carried out as before; the data are given in Table V e ( i n v e r t a s e No. 2) J E and the plot in Figz, ure 6. The same Q 6 proportionality be2v1 J tween concentration of enzyme and num$ 3 5 8 ber of invertons is observed. A third commerCONCENIFiRTIONJOF INVEkTASE ;6/ML cial preparation was FIGURE4. DILUTIOUCURVE FOR checked as in the INVERTASE No. 1 other cases. The results agreed v e r y 7w well with the data on the other invertases. The data for the rate 6m curves are given in Table VI1 and the rate-curve plot in moo Figure 7. The dilution data are found & 300 in TableV (invertase 2" No. 3) and the plot 5 tijin Figure 8. As a further check 2 loo of the uniformity of z cn behavior, the three invertases w e r e TIME I N MINUTES checked over the 30FIGURE 5 . RATECURVES FOR m i n u t e period by INVERTASE No. 2 establishingthe form A 5.0 mg. per ml. of t h e 30-minute B: 2.5 mg per ml. curve for each preparation. The three curves were essentially superposable over t h e e n t i r e period. The r e s u l t s are tabulated in Table VI11 and are plotted in Figure 9. In each case a concentration of preparation was chosen which would correspond to the same number of invertons. The various exp e r i m e n t s show clearly that thenumber of invertons in a g i v e n weight of preparation i s diFIGURE6. DILUTIONCURVEFOR INVERTASE No. 2 rectly proportional to the concentration of the invertase in the preparation. The data also indicate that the method of measurement is accurate and of general applicability to the determination of yeast invertase. Nelson and Hitchcock ( 7 )found that certain invertase prepa,/
It
Vol. 7 , No. 2
rations behaved in an abnormal manner. The abnormality was evidenced by a dropping off of the rate of inversion after approximately 20 per cent of the sucrose had been hydrolyzed in a 10 per cent sucrose solution. They were unable to ascribe this t o any method of preparation of the enzyme concentrate. Fassnacht (3) has elaborated on this work and has given a method for the preparation of abnormal yeast invertase. The authors attempted to prepare an abnormal invertase but have not been able to duplicate Fassnacht's results. They are continuing the work with a view to clearing up the discrepancies.
N
$00
Y 0
[r 0
O
i
2
3
9
5
6
7
8
9
1
TABLEV.
RELATIONBETWEEN INVERTASE AND INVERTON CONCENTRATIONS
INVERTONS PER 25 ML. EXPERIYENTALLY DETERMINED Invertase Invertase Invertase No. 1 No. 2 No. 3 INVERTASE Mg./ml. 6.0 17.04 17.35 17.27 4.0 11.10 11.52 11.39 3.0 8.44 8.72 8.6R 2.0 5.59 5.89 5.83 1.5 4.30 4.47 4.38 1.0 2 83 2.97 2.91 0.76 2.14 2.21 2.18 0.50 1.37 1.43 1.48 0.38 1.03 1.06 1 09 0.25 0.67 0.74 0.71
CONCENTRATION ns
TABLEVI. RATEOF INVERSION BY INVERTASE No. 2 (2.5 mg. of invertase preoaration per ml. used in experiments A and B; 5 mg. per ml. in C) SUCROSE INVERTED A B Av. C Min. Mg. Mu. MR. Mu. 1 35.8 35.8 35.8 71.7 2 76.8 69.1 148 61.4 3 113 107 110 220 4 14s 135 142 287 6 210 215 213 420 8 287 282 285 552 10 363 348 356 681
TABLE VII. RATEOF INVERSION BY INVERTASE No. 3
0
(25 mg. of invertase preparation per ml. used in experiments 5 mg. per ml. in C and D) SUCROSEINVERTEDA B Av. C D Min. Mo. Mo. Mg. MQ. Mn. 35.9 35.9 77 1 35.9 77 2 77.0 77.0 77.0 149 149 3 113 113 113 226 216 4 149 149 287 287 149 215 221 416 6 226 416 287 287 549 549 8 287 10 359 359 359 677 672
A and B ;
Av.
Mo. 77 149 221 287 416 549 675
TABLEVIII. UNIFORMITY OF INVERSION O F SUCROSE BY COMMERCIAL INVERTASE PREPARATIONS Min. 2 4
6 8 10 14 17 20 25 30 a b 0
INVERTASE No. l a A B Av.
INVERTASE No. 2b A B Av.
143 276 404 537 655 880 1044 1190 1423 1607
143 282 415 550 67 1 906 1065 1213 1449 1648
143 287 415 543 660 896 1055 1208 1433 1637
149 282 42 1 544 672 906 1073 1221 1452 1642
146 282 4 18 547 672 906 1069 1217 1451 1645
INVERTASE No. 30 A B Av. 139 267 400 529 652 883 1037 1191 1422 1622
139 272 400
139 270 400
534 652 875 1042 1196 1422 1622
652 532 879 1040 1194 1422 1622
Concentration 5 mg. per ml. Concentration: 4.914 mg, per ml. Concentration, 4.831 mg. per ml.
The preparation of abnormal invertase was attempted in order to test the method as completely as possible. It is apparent from the nature of the invertase method that in order to measure an abnormal invertase accurately it is only necessary to determine a dilution curve €or the preparation in question. If the curve deviates from a straight line it is evident that one is dealing with an abnormal preparation. To measure the inverton content, a dilution should be chosen
March 15, 1935
ANALYTICAL EDITIOE
85
700
21.0 20.0 10.0 18.0 17.0
600
16.0 15.0
so0
,A0
13.0 400
12.0
n
11.0
& 300
100
W
2 z
8.0
E zoo
8.0
"
D cc
I
c1 100
'0
P
7.0
45
6o
,g
I 2
I
3
s
t
6
7
e
D
10
TIME I N MINUTES
FIGURE 7. RATECURVES FOR INVERTASE No. 3
*
LO
4
A , 5.0 mg. per ml. B , 2.5 mg. per ml.
+.o
- 3.0
- 2.0
FIGURE 10. INVERTON NOMOGRAM
V
gz 0
j
Znvarton Nomogram 2. Log I 0 . 0 2 9 8 9 f O . Q Q 9 0
which corresponds to the linear portion of the curve. Any preparation not known to be normal should be checked in this manner. It seems likely that few abnormal preparations will be found in commercial samples.
I
Id
For I values less than 7.50
!
i
g
3
4
CALCULATIONS The number of milligrams of sucrose inverted was calculated by the following formula: S = 2500 X
F -
v
(1)
38
3 7
La not use this Homogram for wZuecs of I 2em than ?so. 42
FIGURE 11. INVERTON NONOGRAM For I values greater than 7.50
FIGURE9. CO~MPARISON OF RATE CURVESFOR SOLUTIONS OF DIFFERENT INVERTASES CONTAINING THE SAME NUMBEROF INVERTONS
where F = fall in polarization after inversion V = drop i n polarization upon corn?'et' inversion
V was calculated by the Hudson formula:
a>
(2)
where B = initial or blank iotation t = temperature in C. The Hudson formula has recently been revised, but the difference is negligible in comparison with the authors' experimental error of 2 Der cent. The two formulas previously given for calculating the number of invertons are easily modified into a form more suitable for rapid calculation. Since the temperature is maintained constant a t 25" C., the Hudson formula reduces to V
=
1.292 B
(3)
INDUSTRIAL AND ENGINEERIKG CHEMISTRY
86
If Formula 3 is substituted into 1 and the resulting eauation into the original inverton formulas, and the resulting equations are simplified, Formulas 4 and 5 are obtained: I
log1 =
F 1.0667 log
+ 1.1690
log I
=
(4)
F
0.8299 B- - 0.4490
LITERATURE CITED
_
for values of Z up to 7.50 (5)
Vol. 7, No. 2
(1) Brown, A. J., J . Chem. Soc., 81,373-88 (1902).
M., "Determination of Hydrogen Ions," 2nd ed., Baltimore, Williams & Wilkins Co., 1922. Fassnacht, H. H., Dissertation, Columbia University, 1930. Gore, H. C., IND. E m . CHEM.,Anal. Ed., 4, 367 (1932). Henri, V., Z. physik. Chem., 39, 194-216 (1901). Michaelis, L., and Menten, M. L., Biochem. Z . , 49, 333-69
(2) Clark, W.
(1913).
Nelson, J. M., and Hitchcock, D. I., J . Am. Chem. Soc., 43,
for values of I greater than 7.50
2632-55 (1921).
Formulas 4 and 5 are easily represented by nomograms, which were constructed by the usual method (Figures 10 and 11). The inverton values are read by aligning the point representing initial polarization with that representing fall in polarization and noting the point a t which the line crosses the inverton scale. ACKNOWLEDGMENT The authors wish to thank Charles N. Frey for his aid and interest in this work.
Nelson, J. M., and Vosburgh, W. C., Ibid., 39, 790-811 (1917). O'Sullivan, C.. and Tomwon. F. W., J . Chem. Soc., 57. 834931 (1890).
Vosburgh, W. C., J . Am. Chem. Soc., 43, 1693-1705 (1921). Weidenhapen. R.. Chem.-Zta.. 58. 185-7 11934). WillstLtter, R., Graser, J., ahd Kuhn, R., 2. physiol. Chem., 123, 1-78 (1922).
Willstiitter, R., and Kuhn, R., Be?., 56,509-12 (1923). RECEIVED October 8, 1934. Presented before the Division of Agricultural and Food Chemistry a t the 88th Meeting of thc American Chemical Society, Cleveland, Ohio, September 10 t o 14, 1934.
Determination of Mercaptans in Hydrocarbon Solvents An Improvement of the Silver Nitrate Method WILLIAM M. MALISOFF'AND CLAUDEE. ANDING,JR., The Atlantic Refining Co., Philadelphia, Pa.
A
PROCEDURE has been described by Borgstrom and Reid (1) for determining mercaptans, involving the formation of silver mercaptide by excess silver nitrate
15 mm. and melting points of 41" to 43" C., respectively. The inorganic reagents were Baker's c. P. analyzed.
PROCEDURE. The tests are performed in glass-stoppered Erlenmeyer flasks. To known (weighed) amounts of mercaptan solutions kept in the dark for as short a period as possible, add 5 to 10 cc. of methanol and an excess of standard 0.005 N silver nitrate. Shake well and add 2 cc. of standard ferric alum indicator. Titrate with 0.005 N ammonium thiocyanate to a very faint pink. Then add a small excess of silver nitrate solution and titrate back again with thiocyanate until the pink appears. The final result is taken as the end point. Constant shaking is necessary throughout, and care must be taken not t o confuse the IN NAPHTHA faint color of the end point with the slight tint due to the presence TABLEI. ANALYSISOF n-hdYL MERCAPTAN of ferric alum. If in doubt, repeat the treatment with excess of FOR MERCAPTAN SULFUR silver nitrate and titration with thiocyanate. MERCAPTAN SULFURFOUND Stock ferric alum solution is made up of 40 grams of ferric Borgetrom-Reid Modified CONCBNTRATION methoda methodb alum and 20 cc. of 6 N nitric acid per 100 cc. It is boiled to ( CALOD.) remove any free nitrogen oxides and then diluted with 3 parts of % % % water to 1 of stock to make up the actual standard indicator. 0.158-0.166 0.196-0.199 0.162
which is titrated back with ammonium thiocyanate, using ferric alum as an indicator. This analog of a known method of chloride determination revealed some difficulties in use, as reported by Malisoff and Marks (3) and in private communications (2). An example of the oscillation in results when the method is applied literally is given in Table I.
0.316 0.738
0.314-0.324 0.730-0.750
0.315-0.399 0.664-0.762
Range of from 4 to 6 analyses (each analysis b Two or more analyses.
=
2 titrations).
At the higher mercaptan concentrations the averages seem to line up better, but one must have more than two analyses per sample and even then may be in doubt. An effort has been made to set up a series of safeguards and a sharper definition of conditions to insure a better degree of reproducibility.
EXPERIMENTAL MATERIALS.The mercaptans and solvents have been described by Malisoff and Marks ( 3 ) . Besides these, phenyl and p-tolyl mercaptans were obtained from the Eastman Kodak Company and had boiling points of 70" to 71" C. a t 1 Present address, Department of Physiological Chemistry, University of Pennsylvania, Philadelphia, Pa.
Using the procedure as outlined, analytical data have been obtained as shown in Table 11. PURITY OF MERCAPTANS. The purity and the stability of the mercaptans were tested in hydrocarbon solution under varying conditions. Amyl mercaptan was the purest and most reliable of those studied. The principal impurities are disulfides which themselves do not affect the analytical method, Mercaptans, especially the aromatic ones, change readily to disulfides by oxidation. The effect is marked in sunlight, and is sufficient to account for discrepancies in analyses carried on before and after exposure to ordinary daylight, The effect also appears to be greater in naphtha solutions than in benzene. Table I11 indicates the effect of exposure to direct ordinary daylight in a southeast window, It is apparent that within a month it is possible to miss all of certain mercaptans in a hydrocarbon solution containing originally about 0.3 per cent of mercaptan sulfur.
