Determination of submicro quantities of ammonia

ANALYTICAL

33, 335-340 (1970)

BIOCHEMISTRY

Determination

of

Submicro

Quantities

ALEXANDER Department

of Biochemistry,

University Received

of

Ammonia

LEVITZKII of

June

California,

Berkeley,

California

9475’0

9, 1969

All of the present methods for ammonia determination require the existen,ce of pure NH, in the assay solution. Distillation of NH, into acid is, therefore, a prerequisite (1) for the analysis of ammonia in the presence of proteins and amino acids. The distilled ammonia is then determined either titrimetrically (2-7) or calorimetrically (8-10). The effective range of both types of methods is in the range of 0.1-1.0 pmole NH, per assay. In this communication a new method is developed that is capable of determining NH, down to 1O-5 M (0.01 PmoleJml assay) in the presence of other nitrogenous compounds such as proteins and amino acids. Thus, no predistillation of the NH, is necessary. Beef liver GDH” catalyzes the reaction: cu-ketoglutarate

f

NH,

+ NADH

+

L(f)-glutamic

acid + NAD

(1)

The K,,, values for the substrates at pH 7.6 were found to be 1.92 x 1O-3 M for glutamic acid, 2.47 X 1O-5 M for NAD, 1.23 X 1O-4M for aKG, 1.8 X 1O-5M for NADH, and 0.057 M for NH,+ (12). As the Km value for NH,+ is very high, it is easily concluded that at ammonia concentrations in the range of S = (l/100) Km (S < Km), the Michaelis-Menten equation reduces to the form:

The initial velocities using saturating (uKG and NADH should be linearly dependent on ammonia concentration. Thus, taking the last statement as a working hypothesis it was decided to follow the oxidation ’ On leave of absence from the Department of Biophysics, The Weismann Institute of Science, Rehovoth, Israel. Recipient of a travel grant under the Fulbright-Hays program, 1968-1970. ’ Abbreviations used : GDH, L(+)-glutamic dehydrogenase ; NAD, nicotineadenine dinucleotide ; NADH, reduced nicotine-adenine dinucleotide; CYKG, cu-ketoglutaric acid (Lu-oxoglutarate). 335

336

ALEXANDER

of NADH by aKG, concentration.

LEVITZKI

both at saturating

levels, as a function

of NH,+

MATERIAL8

Pure GDH in glycerol, ammonia free (10 mg/ml), NADH, and aKG were purchased from Boehringer und Sohne, Germany. All other Chemicals used were of analytical grade. EXPERIMENTAL

A solution of Na,NADH, 2.4 X 1O-3M in 1% was freshly prepared before each experiment. A was prepared by dissolving the material in water 7.6 using 3 N and 0.3 N NaOH. The aKG solution when kept frozen. GDH is diluted in 0.4M potassium phosphate mg/ml.

NaHCO, (1.4 mg,/ml), solution of (wKG, 1 M, and bringing the pH to is stable for one week buffer, pH 7.6, to 0.5

RESULTS

The assay mixture found to match all requirements for reasonable background absorb,ance, high buffer capacity, and linearity of initial rates for 3-5 min is as follows: NADH (2.4 X 10-S M in 1% NaHC08) orKG (1 M, pH 7.6) Potassium phosphate buffer (0.4 M, pH 7.6) Hz0 NH1 or NH*-containing solution GDH (0.5 mg/ml, pH 7.6)

O.lOml

0.05 ml 0.30 ml 0.25 ml

Total volume

0.10 0.20

ml ml

1.0

ml

The assay mixture less the GDH was prepared in 1.5 ml quartz cuvets within a Gilford 2000 equipped with a thermostat at 25”. After preincubation for 5 min, the GDH was added and the rates of NADH oxidation were followed at 340 w on a 0.5 OD full-range scale. Under these conditions the background absorbance is 1.65.3 With each determination of calibration curve, a control without ammonia was included. The net decrease, AA,,,Jmin, namely, initial rate minus control, was measured, and the net AA,,,Jmin versus ammonia concentration was then plotted. The results are presented in Figure 1. Reproducibility of the assay is &5%. It should be noted that the type of ammonium salt used does not affect the results reported in Figure 1. Thus, using (NH,),SO,, am‘The absorption

Gilford is capable of reading of 2.6 OD at, any wavelength.

accurate

OD

changes

above

a background

AMMONIA

0

337

DETERMINATION

0.1 MICROMOLES

0.2 NH,

FIG. 1. Calibration curve for NH3 determinations using glutamic dehydrogenase. The reaction mixture contained: NADH, 2.4 X lo-* M ; (rKG, 0.05 M; potassium phosphate buffer, pH 7.6, 0.12 M; GDH, 100 pg; and NH, as given in the graph. The final volume WSLS1.0 ml. The background (control without NH31 PAW change was 0.3 X 10” OD/min and was subtracted from all values.

or NH&l gave the same values. With this method the glutaminase activity of CTP synthetase was determined (13).

monium

acetate,

Inhibitors L-Glutamic acid and L-glutamine as well as other amino acids and peptides at levels as high as 5 X 1O-3M in the assay solution had no effect on the rate of the reaction measure. Thus, for example, L-alanine, L-tyrosine, H *Gly *Gly *OH, alanine amide, bovine serum albumin (1 mg/ml) , and ,a-amylase (1 mg/ml) do not have any effect on the assay. This is easily explained by the fact that orKG is present at, high concentrations at all times (5 x 1O-2M) and thus occupies the site common to I(uKG and L-glutamate. As L-glutamine does not inhibit the reductive amination of olKG, one can use this assay for determining glutaminase activity. However, one cannot use the GDH assay in the other direction to measure the glutamic acid product of the glutaminase reaction, the reason being that L-glutamine inhibits the oxidative deamination of glutamic acid by GDH (14). ATP and GTP have been shown to be powerful inhibitors of glutamic dehydrogenase (15, 16). Since ADP is an activator of glutamic dehydrogenase in the direction of glutamate synthesis (17)) it, can be used to overcome the inhibition by these compounds, as is seen in Figure 2. Thus, ammonia can bc assayed even in the presence of nucleotides if ADP is added to the assay system. The concentration of ADP to be

338

ALEXANDER

LEVITZKI

MICROMOLES

NH3

Fro. 2. Determination of NE& in the presence of inhibitors and ADP. tion mixture contained: NADH, 2.4 X lo4 M ; ~KG, 0.05 M; potassium buffer, pH 7.6, 0.12 M; GDH, 100 pg; ATP, 1 X 10d4M; GTP, 1 X lo-” 1.35 X 10.*M. The background (control without NH,) AA,,,, change was OD/min and was subtracted from all values.

The reacphosphate

M; ADP, 0.9 X lO-’

included depends on the amounts of ATP or GTP that may be present in the solution. When the concentration of these two inhibitors is worked out, a calibration curve for ammonia in the presence of the nucleotides and ADP can be obtained. The initial rates as a function of ammonia concentration depends on the ratio ADP/total nucleotides. A combination of this type, given in Figure 2, yields initial rates higher than the initial rates in the absence of nucleotides altogether (Fig. l), which means that the inhibitory effects of ATP and GTP were more than overcome completely and the enzyme may even be slightly activated by the excess ADP (17). DISCUSSION

A method for the determination of minute quantities of NH, without the separation of the latter from a mixture has been developed. By using the glutamic dehydrogenase reaction with ‘o;KG as a substrate, one uses GDH as a specific ammonia reagent. The advantage of the assay is that the reagent, GDH, is vulnerable to inhibition by a very small number of specific molecules such as ATP and GTP. In the existing NH, assays (2-10) other molecules such as amino acids, peptides, or proteins interfere when present together with the NH,, thus making isolation of the NH, by distillation (1) inevitable. Finally, the present method is about ten times or more sensitive than the other methods mentioned above because it determines accurately 0.02 .ymole

AMMONIA

DETERMINATION

339

NH, whilethelowerlimit of existingmethods is around0.1-0.2 @mole

NH30

It shddbenoted that,sinceADP activates GDHin the direction of glutamate synthesis, it can be used, in principle, to increase the sensitivity of the method. However, the accuracy of the method decreases, since in the presence of higher ADP concentrations the initial velocities were difficult to obtain because a higher fraction of NH, was consumed per unit time. This problem becomes more serious at low NH, and high ADP. Furthermore, the ‘(background” reaction due to ammonia contamination of reagents becomes a significant fraction of the total ammonia measured at low NH,. Thus, it is recommended to use ADP only when it is necessary to overcome the inhibitory effects of GTP and ATP when present in the sample. SUMMARY

A method for the determination of submicro quantities of ammonia is described. The method makes use of glutamic dehydrogenase as a specific ammonia reagent when applied to the reductive amination of a-ketoglutaric acid. ACKNOWLEDGMENTS The author wishes to thank Mrs. Patricia Spoerl for excellent technical assistance and Dr. D. E. Koshland, Jr., for stimulating encouragement throughout the study. The author wishes to acknowledge the invaluable support of the National Institutes of Health and the American Cancer Society in this research. REFERENCES 1. CONWAY, E. J., in “Microdiffusion Analysis and Volumetric Error,” 5th ed., p. 30. Crosby-Lockwood, London, 1962. 2. HOFFMANN, E., AND SCHMIDT, W., Biochem. 2. 324, 125 (1953). 3. HOFFMANN, E., in “Methods of Enzymatic Analysis” (H.-U. Bergmeyer, ed.), p. 93. Academic Press, New York, 1963. 4. MEISTER, A., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. II, p. 380. Academic Press, New York, 1955. 5. TOWER, D. B., J. Neurochem. 3, 185 (1958). 6. TOWER, D. B., in “Methods in Enzymology” (C. H. W. Hirs, ed.), Vol. XI, p. 88. Academic Press, New York, 1967. 7. RUSSEL, J. A., J. Biol. Chem. 156, 457 (1944). 8. HOFFMANN, E., in “Methods of Enzymatic Analysis” (H.-U. Bergmeyer, ed.), p. 915. Academic Press, New York, 1963. 9. HOFFMANN, G., AND TEICHER, K., Pflanzenerniihrung, Diingung, Bodenkunde 95, (1041, 55 (1961). 10. SUMNER, J. B., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. II, p. 378. Academic Press, New York, 1955. 11. FRIEDEN, C., in “The Enzymes” (P. D. Boyer, H. Lardy, and K. Myrbiick, eds.), 2nd ed., Vol. 7, p. 3. Academic Prem, New York, 1963.

ALEXANDER LEVITZKI 12. STRECHER, H. J., in “Methods in Enzymology” (S. P. Colowick Kaplan, eds.), Vol. II, p. 220. Academic Press, New York, 1955. 13. LEVITZKI, A., AND KOSHLAND, D. E., JR. (in preparation). 14. HARTMAN, S., J. Biol. Chem. 243, 853 (1968). 15. WOLFF, J., J. Biol. Chem. 237, 236 (1962). 16. FRIEDEN, C., Biochim. Biophys. Acta 59, 484 (1962). 17. FRIEDEN, C., J. Bid. Chem. 234, 815 (1959).

and N. 0