V O L U M E 20, NO. 12, D E C E M B E R 1 9 4 8 only through further study. I t is believed, holvever, that this method can bt. used to advantage for the determination of approximate surface tensions of substances Lvhich have a relatively high mrlting point and for those which tend to flow upon heating but do not have sharp and well-defined melting points. Measurements are simplified, as it is not necessary to make them with the material maintained a t a high temperature.
LITERATURE CITED
Figure 1. Pendent Drop of Antimony Trioxide Note orientation of needles
ACKNOWLEDGMENT
1185
formed during The values for lead oxide and lead crystallization chloride were determined in these laboratories by George F. Dasher. The density value used for lead oxide was 8.02 (at 9280c.) w;hichwas kindly furnished by W. X. Harrison (10). This value is Presumabl?. close t o the density value of oxide at its melting point, 888” C. The authors .i?.ishto express their thanks to the Corning Glas? Works, K m b l e Glass co., Libbey-Owens-Ford co., and Pittsburgh ’late ‘lass for and Of glasses in the course of this investigation.
(1) Adam, S . K., “Physics and Chemistry of Surfaces,” p. 161, Oxford, Clarendon Press, 1930. ( 2 ) Andreas, J. M., Hauser, E. H., and Tucker, W.B., J . Phys. Chem., 42, 1001 (1938). (3) Babcock, C. L., J . Am. Ceram. Soc.. 23. 12 (1940). (4) Badger, A. E., Parmelee, C. IT.,and Williams, A. E., Ibid., 20, 325 (1937). ( 5 ) Bartell. F. E., and Davis. J. K., J . Phw. Chem.. 45, 1321 (1941). (6) Bartell, F. E., and Kiederhauser, D.-O., “Corrected Table for Calculation of Boundary Tensions by Pendent Drop -Method,” Am. Petroleum Inst., Project 27, unpublished. ( 7 ) Bartell, F. E., and Zuidema, H. H., J . Am. Chem. Soc., 58, 1453 (1936). (8) Bradley, C. A., Jr., J. Am. Ceram. SOC.,21, 339 (1938). (9) Harkins, W. D., and Jordan, H. F.. J . Am. Chem. Soc., 52, 1751 (1930). (10) Harrison, W. N., National Bureau of Standards, private communication. N.9 and A f O O r e , D. G.9 J . Research A r d . BUT. (11) Harrison, Standards, 21, 337-46 (1938). (12) hIorey, G . w., ”properties of Glass,” p . 210, . ~ M E R I C A NCHEMICAL SOCIETY Monograph, New York, Reinhold Publishing Corp., 1938. December 1Q47. presented beiore the Division of Colloid Chemistry at the 100th Meeting of the . ~ M E R I C A X CHEMICAL SOCIXTY, Detroit, Bfich. RECEIVED
COZYMASE Assay of Diphosphopyridine Nucleotide Preparations SIDNEY GUTCHO
AND
EARL D. STEWART, Schwarz Laboratories, Inc., New York, N . Y
Assay of cozymase preparations for diphosphopyridine nucleotide content by reduction of the diphosphopyridine nucleotide and measurement of the light absorbed at 340 mp by the dihydrocozymase formed was first carried out by Warburg and Christian. With modifications such as those suggested by LePage (3), for example, the method is in very general use. Other methods for estimating coenzyme I have been reviewed by Schlenk and Schlenk (7). A manometric oxygen absorption method has been proposed by Krishnan (2).
B
EC2iUGE of the brief time required for its completion,
the spectrophotometric method is well suited in principle to the assay of cozymase preparations, for both control and standardization purposes. \Then applied repeatedly to the same or similar preparations, however, absorption methods yielded results for the diphosphopyridine nucleotide (DPX) content of cozymase that varied appreciably Kith the F a y in which the reduction was performed. The procedures of Karburg and Christian (8, 9) were studied, therefore, to see n hat factors affected reproducibility and maximum light absorption. The method of assaying cozymase preparations presented here is based on the effects of temperature, time of reduction, and concentration of reducing agent, found in these studies. I t has been applied to materials of widely varying diphosphopyridine nucleotide content with consistent and reproducible results. For study of the conditions necessary for good reproducibility and ma\imum absorption, several grams of cozymase of approximately 507, diphosphopyridine nucleotide content were prepared by a modification of the method of Williamson and Green (IO) and set aside for use in the experiments. All determinations of time and teniperature effects Tvere thus made on the same material, and are reported on an “as is” basis. REAGEYTS
Sodium Hydrosulfite Reagent. A 0.27, solution of sodium hydrosulfite in 1.07, sodium bicarbonate is freshly prepared for each series of determinations by dissolving 100 mg. of sodium hydrosulfite and 500 mg. of sodium bicarbonate in 50 ml. of distilled water in a volumetric flaek. The sodium hydrosulfite, Pure Powder, was obtained from the Amend Drug and Chemical Co. and analyzed 86% sodium hydrosulfite by iodine titration (4).
Sodium Bicarbonate-Sodium Carbonate Buffer, pH 9.7, a ’ sodium bicarbonate and 1% anhydrous solution containing 1% sodium carbonate. APPARATUS
A Beckman quartz spectrophotometer, model DU, with 1.00em. absorption cells, is used. The aerating device is a 25 X 200 mm. test tube which can be stoppered by a two-hole rubber stopper holding a glass-tubing vacuum outlet and a glass-tubing air inlet, the inner end of which extends to the bottom of the test tube and is closed down to a 1-mm. bore, approximately, while the outer end is fitted with a short piece of rubber tubing and a screw clamp. By using Y-tubes several test tubes can be connected to a single vacuum valve. METHOD O F ANALYSIS AND CALCULATION
The following extremely rapid procedure yields mavinium reproducible reduction of the cozymase to dihydrocozymase. Two milliliters of the sodium hydrosulfite reagent are added to approximately 10.0 mg. of cozymase, previously weighed and transferred to a 13 X 100 mm. test tube. (In the experiments on effects of time and temperature, 12.5-mg. samples were assayed.) The tube is immersed immediately in boiling water for 1 minute. During the minute of heating the complete solution of the cozymase is effected rapidly while the initial yellow color (nionohydrocozymase), which appears instantly on adding the reagent to the cozymase, fades out within 30 seconds. Particles of cozymase, which may be caught within the slight foam that forms during heating, should be brought into solution by gentle shaking After 1 minute, the tube is removed from the boiling water bath and immersed in an ice water bath, and enough sodium bicarbonate-sodium carbonate buffer is added nearly to fill the tube. After 30 seconds, the chilled solution is diluted further to 50 ml.
1186
ANALYTICAL CHEMISTRY for the extinction of a buffer solution of the original sample to give the active cozymase content as dihydrocozymase. RESULTS
Effect of Temperature and Time. I n the first experiments the reduction of cozymase was carried out at 38" C. A series of determinations with incubation time as the variable showed a maximum reduction within 15 minutes of incubation, the extent of reduction increasing to a maximum and then decreasing slon-ly as the incubation time I was extended (Figure 1). -it20" C. a maximum which was less than that a t 38" was reached after 851 approximately 35 minutes of incubation. The 120"B same relation between the extent of reduction and the temperature and time of incubation was also noted at 30". The effect of higher temperatures (SO", 60", 100" C.) was then studied; these results, as ell as those for the lower temperatures, are shom-n in Figures 1 and 2. At IOO", heating for 0.5, 1.0, and 1.5 minutes gave the same reduction, which, however, was greater than that obtained a t lower temperatures. That rate of reaction is influenced by temperature was indicated also by the time required for the initial yellow color to disappear; about 10 minutes a t 20", but less than 0.5 minute a t 100" C. Though precautions have sometimes been taken to maintain anaerobic conditions during the reduction of diphosphopyridine nucleotide to dihydrocozymase by sodium hydrosulfite (6, 8, 9), there seems to be no necessity for doing so when the reduction is carried out a t 100" C. with the concentrations of sodium hydrosulfite suggcsted below. Effect of Concentration of Sodium Hydrosulfite. I I I I I I I I I I I 5 1 1 5 2 2 5 3 3 5 4 4 5 5 At 20" C. it, n-as noted that an increase in hydrosulfite concentration increased the reduction of TIME IN MINUTES cozymase and decreased the optimum reduction Figure 2. Extinction-Time Curves for Cozymase at 60" and 100" C. time (Figure 1). At 38" C., the effect of additional hydrosulfite %-as not marked for a single series with the buffer. The alkaline solution of dihydrocozymase n-hich of detcrminations. A t IOO", the range of optimum hydrosulfite results from these steps, or a portion of it (20 nil. are ample) is concentration was determined for reduction a t this temperature. aerated vigorously until the hydrosulfite is oxidized completely. As shon-n in Figure 3, maximum optical density occurs a t 0.15 t o The authors' practice has been to aerate for 5 minutes, read the 0.30% of sodium hydrosulfite. absorption on the spectrophotometer a t 340 mp against a buffer control, aerate for another 3 minutes, and read again a t 340 mp. Effect of Aeration upon Stability. .Aeration time and rate of After the first aeration, incomplete oxidation of the sodium aeration are not critical factors. Aerating for 5 and 8 minutes hydrosulfite, which absorbs well a t 340 mp, is indicated by failure gives similar extinctions, and it seems sufficient t o maintain the to obtain any reading whatsoever, or by a high reading that tends aeration a t a rate which Tvill pa,%a continuous stream of bubbles to decrease slowly. In such a case. a third aeration for 3 minutes can be carried out. through the solution with a minimum amount of foaming. It has been the authors' practice to aerate experiments directly After the optical density of the reduced and aerated solution of after the dilution of the reduced chilled solution to 50-ml. volume. cozymase is obtained, calculation of its dihydrocozymase content Effect of Absorption of Oxidized Form. In some preparations requires the use of a figure for the optical density of a pure an appreciable absorption a t 340 mp exists before reduction by dihydrocozymase solution of known concentration. The molechydrosulfite is accomplished. For the cozymase sample used in ular extinction coefficient for dihydrocozymase has been estithese experiments this correction vas small, only 0.03, compared mated by several observers, and a compilation of some of its to the extinction of 1.05 to 1.10 for the reduced form. published values has been reported by Drabkin ( 1 ) . The authors Unoxidized sodium hydrosulfite also absorbs light at 340 mp; have used the value, e = 5.6 X IO3 established by Schlenk (6,6) hence the second aeration to ensure complete oxidation of hydrofrom measurements on a sample of very high purity. From this sulfite after making the initial measurement. value of E an optical density of 0.84 for a solution of 100 microPrecision of Method. Twenty-one determinations at 100" C. grams of cozymase per ml. is obtained, and used in the calculaon 12.5-mg. samples of the same cozymase preparation gave the tions. optical densities shown in Table I. Time of reduction ranged If the concentration of coenzyme preparation in the reduced from 0.5 to 2.0 minutes, and the hydrosulfite level from 0.15 to solution (250 micrograms per ml. for a 12.5-mg. sample) is known, 0.37,. The diphosphopyridine nucleotide percentage is not the purity of the material in terms of dihydrocozymase is readily corrected for extdnction of the oxidized form. That there is a calculated. Correction of the calculated purity should be made
P
/
I
i
1187
V O L U M E 20, N O . 1 2 , D E C E M B E R 1 9 4 8 ACKNOWLEDGMENT
The authors wish to acknoarledge the mterest of David E. Green in their experiments, and to thank Van R. Potter for his suggestions and exchange of information on cozymase assaying. SUlMMARY
I
s3I 3
3
L
O(
A I Ot5 02 03 04 CONCENTRATION OF SODIUM HYDROSULFITE PERCENT IN 10% SODiLM BICARBONATE
05%
Figure 3. Effect of Sodium Hydrosulfite Concentration on Extinction of Cozymase a t 340 mp and 100" C.
Table I. Optical Density
-4 rapid reproducible method for the spectrophotometric assay of cozymase is presented. The reduction by sodium hydrosulfite in sodium bicarbonate solution is carried out in 1 minute a t boiling water temperature without maintaining anaerobic conditions. Extinction maxima at 340 mp for temperatures down to 20' C. are measurably less than a t boiling mater temperature. For a concentration of 250 micrograms of cozymase preparation per ml., maximum extinction is produced by 0.27, sodium hydrosulfite in 1.0% sodium bicarbonate a t boiling water temperature. The method is applicable to cozymase preparation from 207, diphosphopyridine nucleotide up to nearly theoretical purity levels.
Precision of Assay Method Frequency= D P N , 70 50.0 51.4
1.05 1.08 1 09 1 005
51.9
52.1 52.4 52.6 52.8
1.10
1 105
111
Xiumber of times corresponding optical density was obtained in measurements on same sample. 5
21
linear relation between optical density and concentratlon over the range 100 to 300 micrograms of dihydrocozymase per ml. 1s shown in Figure 4 for two different samples. 9t the 50 to 607, level of the usual cozymase preparation there may be no significant correlation of the diphosphopyridine nucleotide content determined by absorption a t 340 mp with the nitrogen, ribose, and phosphorus analyses, and none was apparent with the two preparations used in these studies. -4 manometric determination (very kindly made by David E. Green) with hydrosulfite in bicarbonate solution gave an assay of 47.57& for compound A (Figure 4) which spectrophotometrically gave an active cozymase content of 51.0%.
,UT Figure 4.
DISCUSSION
The reproducibility of the method of assay when applied to a preparation of somewhat over 50% purity, and the straighttine relationship between optical densities and lower and higher levels of cozymase concentration, indicate that a satisfactory method has been established, applicable to cozymase preparations in the purity range of 20 to 65%. The method, therefore, is suitable for the assay of preparations ordinarily isolated and purified in the laboratory by known procedures. Subsequent to the studies on 50yo material, the method was applied to preparations having 60 to S5Y0 diphosphopyridine nucleotide with equally satisfactory results. Recently, some samples of very pure cozymase (prepared by George Hogeboom, Rockefeller Institute, Xew York, N. Y.) were assayed by the method and found to have extinctions of 0.820 and 0.816 for a concentration of 100 micrograms per ml. [compare with the 0.84 calculated from the molecular extinction coefficient of Schlenk (6, a ) ] . The concentration of hydrosulfite and the technique of reduction described can therefore be applied to high diphosphopyridine nucleotide levels.
DIHYDROCOZYMASE /ml.
Relation between Optical Density and Amount of Cozymase
Reduced to dihydrocozymase i n test solutions for two preparations. Preparation A used t o establish time and temperature relations LITERATURE CITED
Drabkin, D. L., J . Biol. Chem., 157, 563 (1945). Krishnan, P. S., Arch. Biochem., 16, 291 (1948). LePage, G. A., J . Bid. Chem., 168, 623 (1947). Rosin, J., "Reagent Chemicals and Standards," 2nd ed., New York, D. Van Nostrand Co., 1946. ( 5 ) Schlenk, F., J . Biol. Chem., 146, 619 (1942). (6) Schlenk. F., and Gllnther, G., in Bamann, E., and Myrbnck, K., "Methoden der Fermentforschung," Leipaig, Georg Thieme,
(1) (2) (3) (4)
1940. (7) Schlenk, F., and Schlenk, T., Arch. Biochem., 14, 131 (1947). (8) Warburg, O., and Christian, W., Biochem. Z . , 287, 291 (1936). (9) Warburg, O., Christian, W., and Griese, A., Ibid., 282, 157 (1935). (10) Williamson, S., and Green, D. E., J . Biol. Chem., 135, 345 (1940).
RECEIVED July 9,
1948
