oxide was heated in test tubes made of ordinarv elass. which itself shows a faint nitrate reaction with a solution of diphenylamine in concentrated sulfuric acid. Hence a specific test for nitrate in nowdered elass or ceramic materials is indicated. The evanoration residues of waters and soil simples can be tested for the possible presence of inorganic and/or organic nitrogenous compounds by means of pyrolytic oxidation with manganese dioxide. Studies are in progress, aimed at the use of this pyrolytic oxidation as the basis of a new method for the quantitaI -
I
tive determination of nitrogen in organic comnoundn. * LITERATURE CITED
-
Baker, R. H., Barkenbus, C., IND. ENG. CHEM.,ANAL. ED. 9, 135 ,.nnv\ \‘”G’,.
Cheronis N. D., Entrikin, J. B., “Semikicro Qualitative Analysis,” 2nd ed., p. 172, Interscience, New York, 1957. (3) Feigl, F., “Spot Tests in Organic Analysis,” 5th ed., p. 65, Elsevier, New York, 1956. Ibid., p. 93.
Feigl, F., Amaral, J. R., Mikrochim. Acta, in press.
Kaina, G., Reich, A,, Mikrochemie
uer. Mikrochim. Acta 39, 75 (1952). (7) Kainz, G.,Schoeller, F., Mikroehzm. Acta 1954,327. (8) Lassaigne, J. L., Ann. 48,367 (1843). (9) Middleton, H., Analyst 60, 154 (1953). (10) Saltzman, B. E., ANAL. CHEM.26, 1941) (1954). (11) Schanderl, H., Staudenmzyer, T., z. Lebensm.-Untersuch. u.-Forseh. 104, 26 (1956). (12) Smith, F. J., Jones E., “Scheme of
Qualitative Orgakc Andy&,” p. 13, Blackie, London, 1953. (13) Sozzi, J. A,, Niederl, J. B., Mikro-
chim. Acto 1956, 1512; 1957, 496. (14) Wilson, C. L., Analyst 6 3 , 332 (1938). RECEIVEDfor review October 9, 1957. Accepted January 27, 1958.
Simple Microtitrimetric Constant-pH Method for Accurate Enzyme Assays MARTIN SCHWARTZ’ and TERRELL C. MYERS Department of Biologicol Chemistry, University of Illinois College of Medicine, Chicago, 111.
b A simple microtitrimetric-pH procedure is described for rapid, accurate determinations of enzyme activity. The concept which has been employed b y many investigators for enzyme octivity determinations has been modified so that the necessary apparatus is accessible to even the most modest of laboratories. Illustrations of the totol time-course enzyme activity curves are presented for both the hexokinase and acetylcholinesterase systems.
acid groups.
The procedure may be
1 presentaddress, University of Chieago, Chicago, Ill.
illustrated with the hexokinase reaction at neutral pH:
-
+
adenosine triphosphate (ATP) Hexose hexose monophosphate adenosine diphosphate (ADP) H +
+ +
One acid equivalent is liberated for each mole of phosphate transferred from ATP to the hexose. The reaction is followed with the aid of a Beckman Model H-2 p H meter; the pH is kept constant b y the addition of sodium hydroxide from a syringe micrometer
tions in experimental designs. The accuracy and precision of the results ohtained with easily accessible equipment make this modification of interest.
The method has been employed in this laboratory for a detailed study of the kinetics of the hexokinase reaction (6). Several of the kinetic constants of this reaction have been accurately determined with a precision not obtained previously. The essential features of the apparatus are seen in Figure 1. The 1.0-ml. hypodermic syringe which is driven by the micrometric titrator (Micrometric Instrument Co., Cleveland, Ohio) is calibrated for volume delivery by titrab ing a standard acid with a standard base. The standard acid is added from a buret to a 10-ml. beaker containing a capillary magnetic stirrer. The tip of the syringe micrometer is placed in the acid solution and one drop of phenolphthalein is added. The number of micrometer divisions necessary to change the indicator color is recorded. Tahle I shows the results of a calihrai tion. One division indicated on the gage of the micrometer corresponds to a displacement of 0.001 inch. The maximum error of delivery is less than 0.5% in 0.04 ml. One division corresponds Table
I.
Calibration of Syringe Micrometer 1.001N mdium hydroxide Micrometer M1. ,per
HCI Added, MI. HCI, N 4.0 0.0100 Figure 1. Microtitrimetric constant-pH apparatus with constont temperature chamber
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ANALYTICAL CHEMISTRY
6.0 4.0 6.0
0.0100 0.0500 0.0500
Divisions Required 92.5 139.3 464.0 698.0
Divlaan
X 10’ 4.32 4.31 4.31 4.30 Av. 4.31
250
2K
-
150
-
n
:
2 4
100
IO I2 14 16 I8 20 22 24 Minutes
6 8
4
Figure 3. Effect of time on course of acetylcholinesterase reaction 5c
io
2c
30
4.0 ml., pH 7.7, 3 7 ' C., 0 . 0 4 M magnesium chloride, 0 . 0 0 2 5 M potassium phosphate, 0.1M sodium chloride, 0 . 0 0 3 7 3 M acetylcholine ( 1 4.9 pmoles), 0.200 mg. of acetylcholinesterase
Minuter
Figure 2. Effect of time on course of hexokinase reaction 4.0 ml., 3 0 ' C., pH 7.5, 0.02M magnesium chloride, 0.0025M potassium phosphate, 0 . 0 4 M d-glucose, 0 . 0 3 0 mg. of hexokinase 1. 0.006M ATP (24.1 pmoles) 2. 0 . 0 0 4 M ATP ( 1 6.1 p o l e s )
to the addition of 4.31 X nil. Diffusion of the alkali into the solution is negligible. This may be checked by placing the capillary tip in distilled water and recording the p H with time. Each new tip should be checked in this manner. The experiments with hexokinase are carried out in the following manner. All components of the reaction mixture except for the enzyme and d-glucose are pipetted into a 10-ml. beaker of borosilicate glass containing a capillary magnetic agitator. The beaker is inserted into the rubber diaphragm of the Plexiglas temperature chamber. The rubber diaphragm which makes up the surface of the chamber has a circular opening through which small beakers may be conveniently introduced and removed. The diaphragm-beaker connection is mater-tight. The electrodes and the syringe tip are then arranged in the beaker. The pH is adjusted to the preselected value (pH 7.5), the final adjustment being made after adding the enzyme. The reaction is started by adding d-glucose. The pH is kept constant with sodium hydroxide and readings are taken every 15 or 30 seconds. Changes of 5 to 40 divisions per minute are conveniently handled. The division readings of the micrometer are converted to acid equivalents
by multiplying the volume per division by the normality of the sodium hydroxide in the syringe. I n the experiments s h o m in Figure 2, 0.105N sodium hydroxide was used. As the syringe delivered 4.31 X lo-* ml. per division, the number of acid equivalents per division was 0.453 X lo-'. The total volume of sodium hydroxide added in obtaining all points for curve 1 was 0.233 ml. Curve 2 required 0.156 ml. The agreement with theory for the total number of acid equivalents was within 3% (24.1 pmoles of ATP yielded 24.5 X 10-6 acid equivalent; 16.1 pmoles of ATP yielded 16.4 X acid equivalent). The method may be adapted to measure rates a t acid pH where the acidic groups of substrates and products are not completely dissociated. The total acid equivalents liberated a t a desired p H when the reaction has run to completion are determined. This value is compared n-ith that obtained a t pH 7.5 where the acidic groups are completely ionized. The ratio of the value obtained a t pH 7.5 to that a t the desired p H is the correction factor. The rate a t the desired pH multiplied by the correction factor is the true reaction velocity (6). A second illustration of the applicability of the method is given with the enzyme acetylcholinesterase. The reaction a t neutral pH is Acetylcholine .-t choline
+acetate- + H +
Figure 3 shows the results of an experi-
ment with this enzyme. The agreement with theory for the total number of acid equivalents titrated was within 2% (14.9 pmoles of acetylcholine yielded acid equivalent). 15.2 x The method is not limited to enzyme studies. It could, for example, be adapted to measure glycolysis under aerobic and anaerobic conditions. An inert gas such as argon can be passed through the experimental solution to obtain anaerobic conditions. The method has the advantage over manometry (bicarbonate-carbon dioxide technique) of utilization over a much vvider pH range. The rapidity of response is also greater than with manometry, as gas equilibrium problems are eliminated. The sensitivity of the apparatus described yields accurate readings of a magnitude which corresponds to 0.5-p1. lactic acid productions. A partial listing of enzymes which could be assayed n-ith this method is: kinases, phosphorylases, phosphatases, proteolytic enzymes, and coupled dehydroglucose-linked enzyme systems. Any reaction that can be coupled with the enzymes mentioned above could also be followed conveniently. The apparatus allows pK values of compounds to be determined on 5- to 10-pmole quantities. The use of the method is more completely illustrated in a study of the physical properties of hexokinase ( 6 ) . ACKNOWLEDGMENT
The authors wish to thank George Luhr, Department of Physiology, University of Illinois Medical College, for developing the constant temperature chamber, LITERATURE CITED
Glick, D., Biochem. J . 31, 521 (1937). Jacobsen, C. F., LBonis, J., Compt.
rend. trav. lab. Carlsberg. Sir. chim. 27, 333 (1951). Kunitz, AI., McDonald, 11. P., J. Gen. Physiol. 29, 393 (1946). Labeyre, F., Biochim. et Biophys. Acta 22, 72 (1956). Keilands, J. B., Cannon, 31. D., ANAL.CHEY.27, 39 (1955). Schwartz, >I., Myers, T. C., Biochim. et Biophys. Acta (submitted for publication). Wilson, I. B., J. B i d . Chem. 208, 123 (1954).
RECEIVED for review July 2, 1957. iiccepted January 31, 1958. Supported by the Sational Institutes of Health, Public Health Service, grant C-2856, and the National Science Foundation, grant 2191.
VOL. 30, NO. 6, JUNE 1 9 5 8
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