ANALYTICAL RIOCHEMISTRY 53, 309-312 (1973)

A Sensitive Mechanized Determination of ATP -I- ADP

Numerous methods are available for the assay of ATP, e.g., the firefly system (1)) the hexokinase system (2)) and the glyceraldehyde phosphate dehydrogenase reaction (3). The lowest concentration of ATP that can be measured with these methods, if carried out fluorimetrically (4,5), ranges from 0.2-2 PM. We have described a mechanized determination of ATP + ADP using a cyclic enzymatic system (6). With this method concen- trations as low as 0.2 PM ATP + ADP can be determined. We now report an improvement of this system which increases the sensitivity to 6 nM ATP + ADP. The reactions on which this assay is based are given in Fig. 1.

The difference with the earlier described method (6) is the way in which the conversion of ATP into ADP is measured. NADH is consumed not only in the glyceraldehyde phosphate dehydrogenase reaction, but also in the glycerol-3-P dehydrogenase reaction and the lactate dehydrogenase reaction. Therefore, hydrazine used as trapping agent for pyruvate and glyceraldehyde-3-P is left out of the reagents. The combined effect of these changes appears to be a much greater consumption of NADH per cycle than can be expected stoichiometrically. The conditions are chosen such that the oxidation rate of NADH is proportional to the concentration

NADH+H+ NAD+

3-Eglycerate l&F&glycerate L-J 3+glyceraldehyde

ATP ADP

&mQyruvate I

dihydmyacetone-f

L

NADHtH+ NADHtH+

NAP NAD+ =i

lactate glyceroWp

FIN. 1. Reaction scheme for the determination of ATP + ADP. 309

Copyright @ 1973 by Academic Press, Inc. All rightg of reproduction in any form reserved.

310 SHORT COMMUNICATIONS

of ATP + ADP (5-50 nM). A high reproducibility is obtained by mech- anization of the method.

Preparation of samples and standards, and calculations were described earlier (6). The optimal composition of the reagent solutions is given in Table 1. All reagents were obtained from Boehringer, Mannheim, W. Germany. For optimal results, it was necessary to free the enzyme mixture from most of the (NH,),SO, by filtration over Sephadex G-25 coarse. Twelve ml of the enzyme mixture (containing 20% more of each protein than indicated in Table 1) was put on a column and eluted with Tris- HCl, pH 7.4, at 20°C (3 ml/min) . The first 9 fractions of about 1 ml were pooled and the final volume was brought to 15 ml with the elution buffer.

The values obtained with the cyclic enzymatic system showed a stan- dard deviation of 2.5 and 0.8% at 12.5 and 25 nM ATP + ADP, respec- tively (n = 10, see Fig. 2). It is now possible to determine the amount of ATP + ADP in 104 human blood lymphocytes [ 1 mM NH,OH lysates (8) 1. We found that all the commercially purified enzymes can be sub- stituted by ‘Laldolase paste” (7) without changing the sensitivity or the reproducibility. “Aldolase paste” was prepared from rabbit muscle as described by Racker (7). A solution of the crude enzyme mixture contain-

TABLE 1

Composition of Reagent Solutions

Reagent lines Composition

I (1.6 ml/min) 25 mM Tris-HCI buffer (pH 7.4, 20°C) 50 /.LM NADH

II (0.15 ml/min) 25 mM Tris-HCl buffer (pH 7.4, 20°C) 5 2 mM P-enolpyruvate

17 mM 3-P-glycerate

66 mM KC1 20 mM MgSOl

III (0.23 ml/min)b 25 mM Tris-HCl buffer (pH 7.4, 20°C) 6 mg/ml albumin 0.32 mg/ml3-P-glyceraldehyde dehydrogensse (EC 5.3.1.9)

0.33 mg/ml 3-P-glycerate kmase (EC 2.7.2.3) 0.34 mg/ml pyruvate kinaae (EC 2.7.1.40) 0.05 mg/ml lactate dehydrogenase (EC 1.1.1.27)

0.006 mg/ml triose-P-isomer&se (EC 5.3.1.1) 0.075 mg/ml glycerol-3-P dehydrogenase (EC 1.1.1.8)

= Reagent I and III were freshly prepared with doubly distilled water containing 1 drop of Levor IV wetting agent (Auto-Analyzer, No. T21-0332) and 0.5 mM EDTA. Reagent II could be kept frozen at -20°C for several months.

b (NH&SOI was removed from this mixture by filtration over Sephadex G-25 coarse. Dilution of the enzyme concentration during this procedure was taken into account

(see text).

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123-4567 8 9 10 - 11 12 13 14

time I

FIG. 2. Recording of a series of ATP + ADP determinations. (1) ‘Base line’ caused by reagents I and II (Table l), (2) decrease of the fluorescence caused by con- sumption of NADH after introduction of reagent III (compensated by opening of the lightslit), (8) 2 water samples, (4) pilot sample (31 nM ATP), (6) 2 water samples, (6) pilot sample (31 nM ATP), (7) 2 water samples, (8) 5 standard samples (6-31 nM ATP), (9) 4 water samples, (10) 10 standard samples (12.5 nM ATP), (11) 11 standard samples (25 nM ATP), (22) 2 water samples, (IS) 6 min continuous sampling of 19 nM ATP, (14) 2 water samples.

ing approx 100 mg protein/ml was incubated at 37°C for 30 min in order to inactivate contaminating phosphofructokinase. In order to reduce spontaneous NADH consumption of this preparation, it was treated with 10 mg active carbon powder/ml. The carbon was eliminated by centrif- ugation and subsequent filtration. The filtrate was then diluted 4 times with 25 mM Tris-HCl buffer (pH 7.4,20”(Z), containing 4 mg albumin/ml. In this solution the activity of the 6 single enzymes was: S-P-glyceralde- hyde dehydrogenase, 7 IJ/ml; 3-P-glycerate kinase, 42 U/ml; pyruvate kinase, 83 U/ml; lactate dehydrogenase, 145 U/ml; triose-P isomerase, 22 U/ml; glycerol-3-P dehydrogenase, 1.4 U/ml.

Using this solution as reagent II (Table 1) , we found no interference of 300 ,UM glucose, 300 nM glucose-l-P, 300 nM glucose-6-P, 10 nM glucose- 1,6-P,, 15 nM 2,3-P3-glycerate, 2.5 ,UM glycerol, 4 PM pyruvate, 150 nM AMP, or 3 mg/ml human AB-serum. Comparing the previously described cyclic determination (6) and this new method, we found less than 5% difference with erythrocyte lysates if the quenching of the fluorimetric signal by haemoglobin was taken into account. With leukocytes, less than 10% difference was found. ATP added to these lysates was recovered quantitatively. This indicates that the determination is specific for ATP + ADP in lysates of human erythrocytes or leukocytes.

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ACKNOWLEDGMENT

This study was supported by financial aid from the Netherlands’ Organization for the Advancement of Pure Research (Z.W.O.), The Hague, The Netherlands (grant No. 91-5).

REPKRENCES

1. STREHLER, B. L., AND MCELROY, W. D. (1957) in Methods in Enzymology (Colo- wick, S. P., and Kaplan, N. O., eds.), Vol. 3, p. 871, Academic Press, New York.

2. KORNBERG, A. (1959) J. Biol. Chem. 182, 779. 3. THORN, W., PFLEIDERER, G., FROWEIN, R. A., AND Ross, I. (1955) Pfliigers Arch.

Ges. Physiol. 261, 334. 4. LEESE, H. J., AND BRONK, J. R. (1972) Anab. B&hem., 45,211.

5. GREENGARD, P. (1962) in Methoden der Enzymatischen Analyse (Bergmeyer, H. U., ed.), p. 553, Verlag Chemie, Weinheim.

6. Loos, J. A., AND PRINS, H. K. (1979) Biochim. Biophys. Acta 201,185. 7. RACKER, E. (1947) J. Biol. Chem. 167, 843. 8. Roos, D., AND Loos, J. A. (1970) Biochim. Biophys. Acta 222, 565.

J. A. Loos RINEKE C. H. VAN DOORN D. Roos

Central Laboratory of the Netherlands Red

Cross Blood Transfusion Service P.O. Boz 9190

Amsterdam, The Netherlands Received September 1,197.S; accepted November 14,197d