Acta Physiologica 83. (1995)

3. szám - PHYSIOLOGY-PATHOPHYSIOLOGY - V. Leskovac-Svetlana Trivic-B. M. Anderson: A novel spectrophotometric method for the enzymatic determination of NAD+ and NADH

Acta Physiologica Hungarica, Volume 83 (2), pp. 243 - 248 (1995) A novel spectrophotometric method for the enzymatic determination of NAD+ and NADH V. Leskovac,* Svetlana Trivic,** B. N. Anderson*** * Faculty of Technology, Bulevar Cara Lazara 1, 21000 ** Faculty of Sciences, Novi Sad, Yugoslavia, and *** Viriginia Polytechnic Institute and State University, Blacksburg, USA Received November 30, 1994 Accepted May 24, 1995 The theory and practice of a novel spectrophotometric method for the enzymatic determination of NAD+ and NADH is described. The method can not discriminate between NAD+ and NADH, but determines the concentration of the sum of both nucleotides. The method is based on the bleaching of p-nitroso-N,N-dimethylaniline (NDMA) (E440 nm = 35400 M 'em ') with NADH, in the presence of ethanol and yeast alcohol dehydrogenase, under the conditions of enzymatic cycling (ethanol > NDNA > NAD/H). The initial rates of -NDMA bleaching are proportional to the concentration of NAD+ or NADH, in a broad range from 10 nM to 100 p.M. Keywords: NAD + , NADH, spectrophotometry, enzymatic determination, nucleotides, ethanol Quantitative determination of NAD+ (ß-nicotinamide adenine dinucleotide) and NADH (ß-nicotinamide adenine dinucleotide, reduced form) in micromolar concentrations is usually performed enzymatically. Detection and quantitation of NADH is based on its strong absorption band at 340 nm (e = 6200 M-1cm_1) and of NAD+ on its absorption band at 260 nm (e = 18000 M-1cm-1); for the quantitative determination, NAD+ is usually reduced enzymatically to NADH [1]. A 10 to 100-fold increase in sensitivity is obtained by measuring the concentrations of NADH fluorimetrically; excitation of NADH at 340 nm affords a strong fluorescence at 430 nm [2, 3]. Further increase in sensitivity is obtained by the method of enzymatic cycling; this method is reported to increase the sensitivity of determination of coenzymes into the picomolar range [1, 4]. The disadvantage of this method is the high cost of chemicals [1]. Correspondence should be addressed to Vladimir Leskovac Faculty of Technology, Bulevar Cara lazara 1 21000 Novi Sad, Yugoslavia 0231-424X/95/S 4.00 © 1995 Akadémiai Kiadó, Budapest

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