Edition
Analytical Volume 3
JANUARY 15, 1931
Number 1
Estimation of DiastaticIEnzyme Preparations’ Taka-Diastase, Malt Diastase, andiPancrea tic Diastase Taichi Harada DEPARTMENT OB BIOCAEMISTRY, NEWY O R K POST-GRADUATE MEDICAL SCHOOL AND HOSPITAL, NEW YORK,N. Y.
T
HE present method is a m o d i f i c a t i o n of Lintner’s p r o c e d u r e
(s), being based in principle
A method is described for the estimation Of diastatic power of taka-diastase, malt diastase, and pancreatic diastase. A table for each shows Lintner’s value against observed starch titrations. The starch s o h tions are buffered with acid Potassium phthalate and sodium hydroxide solution to give more accurate resuits than have been PreviouslY d ~ c r i b e d . A new standard unit is offered and calculated in terms of Lintner’s value. The starch titration is tabulated to show the corresponding glucose content,
uponthe production of ad&&e quantity of r e d u c i n g s u g a r by the action of a d i a s t a s e u p o n a definite amount of Lintner’s starch solution under definite conditions. Ling (2) has suggested a convenient modification of Lintner’s method, but his calculation equation does not represent the true value since the quantity of enzyme used and the amount of sugar produced are not exactly proportional. Recently Oshima (4) proposed a table setting forth the relationship between Lintner’s value (L. V.) and the amount of starch solution digested by the enzyme which is required to just reduce 5 cc. of Fehling’s solution. Lintner’s value was obtained experimentally in a series according to the original Lintner method with different strengths of diastatic solutions. I n practice the calculation values from Oshima’s table do not agree with the right Lintner’s value when examined with a different percentage of each enzyme solution of malt and taka-diastase since his starch solution was not buffered (except with a dilute sodium hydroxide solution), The pH value of his starch solution might vary with different percentages of enzyme solution, The isoelectric point of taka-diastase has been found to be at pH 7.0. It is very necessary to know from time to time the cornparative strength of taka-diastase (Aspergillus oryzae diastase), malt diastase, and pancreatic diastase for the food and textile world as well as for pharmaceutical purposes in the industrial laboratory. It is also essential to establish a new standard unit for these enzymes under definite conditions. Therefore, the author presents the following method, tables, and unit devised for these three enzyme preparations to the end that more convenient and accurate results may be obtained. Two other typical methods, those of Wohlgemuth (7) and of Sheman, Kendall, and Clark (6),are mentioned in the literature, but these are not convenient for industrial laboratory Purposes for many reasons. &ace does not Permit a discussion of them here. 1 Received
June 3, 1930.
Acid Potassium Phthalate and Sodium Hydroxide Buffer Starch Solution
Twenty grams of dry soluble s t a r c h (6) were suspended in a little cold water in a liter beaker, then about 500 cc. of boiling water were added, and the whole boiled for 2 minutes. After cooling, 250 cc. of acid p o t a s s i u m phthalate and 0.2 M sodium hydroxide solution-150 cc. for taka-diastase, 162.5 cc. for malt, and 227 cc. for pancreaticwere added, followed by sufficient distilled water to make 1 liter. One hundred cubic centimeters of this should not completely reduce 5 cc. of Fehling’s solution. This solution should be pH 5.2, as tested with chlorophenol red and methyl red. For the enzyme of Aspergillus orgzae (taka-diastase) this is the optimum hydrogen-ion concentration under the conditions of this method, but for malt diastase and pancreatic diastase, it should be set a t pH 5.3 and 6.0, respectively (1). Enzyme Solution
Exactly 0.05 to 0.5 gram, according to its strength, of finely powdered diastase is weighed Out. This is first suspended in 3 to 5 CC. Of distilled Water in a Small glass mortar, then completely dissolved by further addition of water and introduced into a 500-cc. flask by washing with distilled water until the 500-CC.mark is reached (0.01 to 0.1 per cent solutions)* Procedure and Unit
One hundred cubic centimeters of the 2 per cent Lintner’s starch solution were introduced into a 150-cc. Erlenmeyer flask by means of a 100-cc. pipet. It was kept in an Ostwald thermostatic bath at 37” c. ( ~0.050) t until the starch ~ 0 1 ~ tion reached that temperature (about 30 minutes) after which 10 cc. of the enzyme solution were added and digestion carried on for exactly 30 minutes at 37” C. At the end of this time, 10 cc. of 0.25 M sodium hydzoxide solution were added with shaking to stop further reaction. It was removed from the bath and cooled to room temperature. The total starch solution, therefore, should be 120 cc. Fehling’s solution is standardized by treating 5 cc. of it with 0.5 per cent
Vol. 3, No. 1
ANALYTICAL EDITION
2
pure glucose solution drop by drop, while boiling, until the Fehling's solution becomes colorless. Five cubic centimeters of the glucose should be required. Some of the digested starch qolution is transferred to a buret and gradually run into 5 cc. of the tested Fehling's solution in a test tube. The tube, held in a wire basket, is immersed in boiling water from time to time for about 10 minutes, and then the color is examined. This is continued until the solution just loses its blue color. Thus an approximate volume of the starch solution is ascertained, then the exact volume by repeating the process. When the experiment is carried out with 10 cc. of 1 per cent enzyme solution and it takes just 10 cc. of the starch solution to reduce the standardized Fehling's solution, this is taken as a new standard unit. When 0.12 gram of Lintner's enzyme preparation digested 10 cc. of 2 per cent starch solution so that just enough sugar was produced to reduce exactly 5 cc. of Fehling's solution under definite conditions, he designated this as IO0 units. It has been customary to refer to this as Lintner's value (L. V.). The procedure described by the author is equivalent to 120 L. V. It is highly desirable, from a commercial standpoint, to retain the L. V. terminology. The author offers a more satisfactory method for arriving at these values. Calculations
The enzyme solutions were prepared as follows: A sample of taka-diastase, weighing 0.118 gram and free from sugar,
was dissolved in 500 cc. of distilled water. This serves as a stock solution of 0.236 per cent strength. Portions of this were further diluted with increasing amounts of distilled water to make solutions of various percentages. Each solution was treated as described above. The data are given in Table I. Table I-Lintner's
No. STOCK SOLN. SOLN.
cc. 100 50 30 25 20 10 10 10 5
Values against Starch Titration STARCH SOLN. CALCD. REQUIRED FOR VALUE FOR ADDED STRBNQTH OF 5 cc. FEHLING'S 1% ENWATERSTARCH SOLN. SOLN. ZYME SOLN. cc. % cc. L. v. 0 0.236 3.60 600 50 0.118 5.25 300 70 0.0708 7.40 180 8.50 150 75 0.059 80 0.0472 10.00 120 90 0.0236 15.76 60 190 0.0118 25.00 30 490 0,00472 40.00 12 495 0.00236 67.50 6
By definition, if 10 cc. of digested starch solution reduce 5 cc. of Fehling's solution, its Lintner's value is 120 provided its strength is 1 per cent. However, as shown in Table I, solution No. 5 of the enzyme solution is only of 0.0472 per cent strength. Therefore, for 1 per cent of the diastase
solution, the true diastatic power of the sample diastase is L. V.:true L. V. = per cent of so1ution:l per cent 120:X = 0.0472:l
X
= 2542
Table 11-Lintner's Values of Diastatic Enzyme Preparations against the Starch Titration CORRESPONDING CORRESPONDING TAKAMALT PANCREATI c AMOUNT OF STARCH MALT PANCREATICAMOUNT OF TAKASTARCH TITRATION DIASTASE DIASTASE DIASTASE GLUCOSE GLUCOSE TITRATION DIASTASE DIASTASE DIASTASE L. L. v. L. v. cc. L. I.. v. % L. cc. % 56.5 19.25 51a 343a 257a 4.30 0.1380 45 56 50 19.50 0 . 50005 280 224 332a 5.00 55 19.75 213a 0.4750 260 5.25 300a 49 54 0.1360 20.00 43.5 257.3a 5.30 53 20.25 20s 0.4500a 271a 240 5.50 47 0.1330 20.50 52 0.4330 226 19s 5.75 254 20.75 51 0.4160 213a 193 240 6.00 46 0.1310 40 50.5 21.00 0.4000a 187 225a 205 6.25 21.25 0.3880 180a 214 197 6.50 44 49a 0.1280 21.50 0.3760 190 174 6.75 203 21.76 169 0.3625 181 192 7.00 0.1260 47.5 22.00 37.6 165 176 7.26 182 0.1250a 22.25 0 . 3500a 7.35 42 360 22.50 180a 7.40 44.5 0.1225 34.5 23,OO 171.Sa 7.45 0.1210 40 23.50 0,3380 168 154 176 7.50 0.1190 42 38 24.00 32 0.3280 162 150 169 7.75 0.1170 40 24.50 0.3185 147 158 162 8.00 0.1160 36= 39.5 30a 25.00 0.3075 143 153 155 8.25 28.6a 25.60 0.3000" 146 140 150a 8.50 0.1140 34 37 28 26.00 0.2926 136 143 142 8.75 360 26.50 0.2835 132 13Sa 137 9.00 0.1120 32 34.5 26 27.00 0.2750 129 133 133 9.25 27.50 0.2680 126 129 * 9.50 129 0,1090 30 32.6 25 28.00 0.2615 123 124 9.75 124 28.50 0.2550 120a 120a 10.00 120a 0.1070 30.5 23 29.00 0.2500" 10.20 28 29.50 0,2480 114 117 114 10.25 0.1030 28.5 27 22a 30.00 0.2430 114 111 10.50 111 30.50 0,2370 111 109 10.75 108 0.1010 26 27 21 31.00 0,2320 108 106 11.00 104 0.1000a 25 31.50 0.2275 104 103 11.25 100 0.0980 19.7 32.00 0.2230 101 97a 100 11.50 24a 32.50 0.2180 99 94 98 11.75 0.0970 23 23 33.00 96 0.2150 12.00 900 96 18 33.50 94 0.2115 88 93 12.25 22 0.0960 22 34.00 0.2070 92 90 12 50 85 17 34.50 0.2030 90 88 12.75 82 0.0935 35.00 0.2000" 88 86a 80 13.00 20 20 16 36.00 0.1975 86 84 79 13.26 0.0900" 15a 36.50 0.1925 85 82 77a 13.50 0.0870 14.5 37.00 0,1880 s4a 80 75 13.75 0.0850 17 18 38.00 0.1850 81 78 14.00 73 0.0836 17.2 39.00 0.1825 80 70 76 14.25 0.0820 15 12 40.00 0.1775 78 69" 74 14.50 0.0780 16 41.00 T1 0.1760 77 67 73 14.75 13 0.0750a 42.00 0.1735 75 65 71 15.00 0.0735 14 100 43.00 0.1710 74 63 69 15.25 12" 43.50 73 0.1680 62 68 15.50 0.0725 44.00 0.1670 72 BOa 15.75 0.0715 10.5 9 45.00 0.1625 71 65 59 16.00 0.0700 12 46.00 0.1615 70 57.5 16.25 0.0685 47.00 0.1590 68 62a 57 16.50 8 0.0670 8 48.00 0.1570 67 16.75 0.0660 49.00 66 0.1550 60 17.00 54 7 0.0645 10" 7.5a 50.00 0.1525 65 17.25 0.0630 51.00 0.1520 64 17.50 5s 52 4.8" 0.0620 7 52.00 0 . 1500a 62 17.75 0.0600 53.00 0.1480 61 56 50 18.00 0.0590 54 00 0.1470 59 18.25 0.0580 6.8 8.6 55.00 0.1450 54 60" 48" 18.50 0.0600a 65.00 68 53 18.75 Experimental data. 0.1415 57 62 46 19.00
v.
v.
v.
'
INDUSTRIAL A N D
January 15, 1931
ENGINEERING CHEMISTRY
I n order to obtain the Lintner’s value for 1 per cent enzyme solution for any given solutions, multiply 2542 by per cent of solution. Table I1 represents complete data for the three diastasesnamely, taka-diastase (A), malt diastase (B), and pancreatic
-
55
60
>- .
45
40
TKA-DIASTASE
hULTqlffiTA6E
PmTICDlASTA&
-%--*-
3s
3
30
0 2 . 5 20
15. IO
3
The relations between starch titration and calculated Lintner’s values to 1 per cent enzyme solution of these three enzymes are also given in the figure. In testing taka-diastase, the data of Table I may also be used a t 50” C. where digestion is carried on under the same conditions except that the starch solution is buffered a t pH 5.4 and is made by using 250 cc. of 0.2 M acid potassium phthalate and 177 cc. of 0.2 M sodium hydroxide solution. The calculation is not altered. I n routine practice it is possible to be more economical and use more dilute buffer solutionsthat is, one-fifth of the amount of each buffer solution for the starch solution. Ten cubic centimeters of 0.2 M sodium hydroxide solution instead of 0.25 M sodium hydroxide are sufficient to stop further reaction. The last column in Table I1 gives the percentage of pure glucose which corresponds to the observed starch titration. Literature Cited
1-
0
20
40
60
00
100
120
140
180
I
180
I
200
L. v.
I
220
I
1
1
240 260 l&2
I
I
I
300 320 340 L O
Starch Titration against Calculated Values i n Terms of L. V. t o Per Cent Enzyme Solution
(1) Clark, “The Determination of Hydrogen Ions,” Williams and Wilkins, 1928. (2) Euler, “General Chemistry of the Enzymes,” p. 290, Wiley, 1912 ~. ._
diastase (C)-where the observed titration values were obtained by the above described method. The references (a) data Obtained by repeating with represent various Der cent enzvme solutions. Unmarked numbers are interpolated data obtained from the curve.
(3) Lintner, J . prakl. Chem., 34, 386 (1886).
i:; , “ h “ , 4 i ~ ~ ~ ; ’ ~ ~ D , k ~ a ~ d c ~ ~ ~ ,
3a, 1082 (1910). (6) Waksman and Davison, “Enzymes,” p, 154, Williams and Wi]kins, 1926 _ .__
(7) Wohlgemuth, Biochem. Z., 9, 1 (1908).
Quantitative Determination of Potassium b y Sodium Cobaltinitrite Method’ Pierre J. Van Rysselberge STANFORD UNIVERSITY, CALIF.
HE precipitation of potassium by the sodium cobaltinitrite reagent, studied by de Koninck ( 5 ) ,Gilbert (4), Adie and Wood ( I ) , Cunningham and Perkin (3)) Vurtheim ( 7 ) , and others, has recently been thoroughly investigated by Bonneau ( 2 ) . The difficulties of the method are reviewed in some of the bulletins of the U. S. Department of Agriculture (8). Bonneau’s work confirms the results obtained by Adie and Wood, who showed that the formula of the potassiunisodium cobaltinitrite is KzNaCo(NOz)a.nHsO. According to Bonneau, a constant composition of the precipitate (disregarding the amount of water of hydration) is obtained only if the ratio of the concentration of sodium to that of potassium is larger than 25. Below that ratio the molecule of potassium-sodium cobaltinitrite contains more potassium and less sodium than indicated by the formula given above, the composition tending towards the limiting formula KsThe the temperature at which N~CO(NO~)~.~H ~ Ohigher . precipitation takes place, the higher the amount of potassium in the molecule. The formula found by Vurtheim is K1.5Nal.6Co(NOz)~.nHz0, a result that the work of Bonneau contradicts. Gilbert gives the same formula as Viirtheim. The results of Adie and Wood and those of Bonneau show that the number of water molecules attached to each molecule of KzNaCo(NOz)6is usually one. According to Adie
T
1 Received
July 30, 1930.
and Wood, drying the precipitate below 130” C. has a negligible influence on the final composition. After a long series of determinations of potassiucm in mixed solutions of potassium and sodium chlorides, the writer was able to draw a few interesting conclusions in regard to the sodium cobaltinitrite method, which are presented here as a complement to the work of Bonneau. The reagent was the same as that used by Adie and Wood (1). Bonneau (9) used cobalt nitrate instead of cobalt acetate, The writer usually had to determine small changes of concentration of potassium resulting from electrolytic migration (6). Those changes were determined by comparing the unknown samples with samples of the original solution for which the amount of potassium was known. It was necessary to use samples having equal volumes and to add to all of them the same amount of reagent. If, moreover, the time during which the precipitates were allowed to settle was exactly the same for all the samples to be compared, satisfactory results could be obtained. The influence of the time of settling seems to have been overlooked by all the authors who investigated this method, with the exception, perhaps, of Vurtheim. He allowed the precipitates to settle for 18 hours, a time after which no increase of weight of the precipitates could be detected. Adie and Wood let the precipitates settle overnight. Bonneau few hours.” speaks of
