oxide was heated in test tubes made of ordinarv elass. which itself shows a faint

tive determination of nitrogen in organic comnoundn.

uer. Mikrochim. Acta 39, 75 (1952). (7) Kainz, G., Schoeller, F., Mikroehzm.

I - I - * nitrate reaction with a solution of di- phenylamine 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 man- ganese dioxide.

Studies are in progress, aimed at the use of this pyrolytic oxidation as the basis of a new method for the quantita-

(3)

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.

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

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 63 , 332 (1938).

RECEIVED for 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 pro- cedure is described for rapid, accurate determinations of enzyme activity. The concept which has been employed b y many investigators for enzyme oc- tivity determinations has been modi- fied 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.

illustrated with the hexokinase reaction at neutral pH: Hexose + adenosine triphosphate (ATP) - 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 by the addition of sodium hy- droxide from a syringe micrometer

acid groups. The procedure may be tions in experimental designs. The ac- curacy and precision of the results oh- tained with easily accessible equipment make this modification of interest.

1 present address, University of Chi- eago, Chicago, Ill.

Figure 1. Microtitrimetric constant-pH apparatus with con-

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 deter- mined with a precision not obtained pre-

The essential features of the appara- tus 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 phenol- phthalein 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 maxi- mum error of delivery is less than 0.5% in 0.04 ml. One division corresponds

viously.

Table I. Calibration of Syringe Micrometer

1.001N mdium hydroxide HCI Micrometer M1. ,per

Added, Divisions Divlaan MI. HCI, N Required X 10’ 4 . 0 0.0100 92.5 4.32 6 .0 0.0100 139.3 4.31 4 . 0 0.0500 464.0 4.31 6 . 0 0.0500 698.0 4.30

Av. 4.31 stont temperature chamber

1150 ANALYTICAL CHEMISTRY

250

2 K

150 - -

n

: 100 4

5c

io 2c 30 M i n u t e r

Figure 2. hexokinase reaction

4.0 ml., 3 0 ' C., pH 7.5, 0.02M magnesium chloride, 0.0025M potassium phosphate, 0 .04M d-glucose, 0 .030 mg. of hexokinase 1. 0.006M ATP (24.1 pmoles) 2. 0.004M ATP ( 1 6.1 p o l e s )

Effect of time on course of

to the addition of 4.31 X nil. Dif- fusion of the alkali into the solution is negligible. This may be checked by placing the capillary tip in distilled water and recording the pH 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 boro- silicate glass containing a capillary mag- netic agitator. The beaker is inserted into the rubber diaphragm of the Plexi- glas 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 con- stant with sodium hydroxide and read- ings are taken every 15 or 30 seconds. Changes of 5 to 40 divisions per minute are conveniently handled.

The division readings of the microm- eter are converted to acid equivalents

2 4 6 8 IO I2 14 16 I8 20 22 24 M i n u t e s

Figure 3. Effect of time on course of acetylcholinesterase reaction

4.0 ml., pH 7.7, 37 ' C., 0 .04M mag- nesium chloride, 0 .0025M potassium phosphate, 0.1 M sodium chloride, 0 .00373M acetylcholine ( 1 4.9 p- moles), 0.200 mg. of acetylcholine- sterase

by multiplying the volume per division by the normality of the sodium hy- droxide in the syringe. In the experi- ments 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 meas- ure 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 pH when the reaction has run to com- pletion 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 ob- tained a t pH 7.5 to that a t the desired pH is the correction factor. The rate a t the desired pH multiplied by the cor- rection factor is the true reaction ve- locity (6).

A second illustration of the applica- bility of the method is given with the en- zyme acetylcholinesterase. The reac- tion 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 15.2 x acid equivalent).

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 ob- tain anaerobic conditions. The method has the advantage over manometry (bi- carbonate-carbon dioxide technique) of utilization over a much vvider pH range. The rapidity of response is also greater than with manometry, as gas equilib- rium 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 de- hydroglucose-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 com- pletely illustrated in a study of the physical properties of hexokinase ( 6 ) .

ACKNOWLEDGMENT

The authors wish to thank George Luhr, Department of Physiology, Univ- ersity 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., Bio- chim. et Biophys. Acta (submitted for publication).

Wilson, I. B., J. Bid . Chem. 208, 123 (1954).

RECEIVED for review July 2, 1957. iic- cepted 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 1958 1151