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

244 V. Leskovac et al. In 1971, Dunn and Bernhard discovered a novel substrate of liver alcohol dehydrogenase - /)-nitroso-Ai, A'-dimethylaniline [5, 6]. Recently, we have found that NDMA is also a good substrate of yeast alcohol dehydrogenase [7]. NDMA has excellent spectral properties, with a strong absorption band at 440 nm (e = 35400 M_1 cm“1, pH-independent) and a low absorbancy at 340 nm (e = 1000 M“1 cm-1) [5, 7]. For this reason, NDMA is uniquely suited for the enzymatic determination of NAD+ and NADH. In this communication, we have described a novel spectrophotometric method for the enzymatic determination of NAD+ and NADH, with NDMA and yeast alcohol dehydrogenase. The method is effective in a broad range of coenzyme concentrations, from 10 nM to 100 p.M. Materials and methods Yeast alcohol dehydrogenase (EC 1.1.1.1), NAD+ and NADH of the highest grade purity, were obtained from Boehringer GmbH, Mannheim (Germany). p-Nitroso-7V,lV-dimethylaniline was obtained from Aldrich-Chemie GmbH, Steinheim (Germany). All other chemicals were of analytical grade purity, obtained from commercial sources. The concentration of enzyme protein in solution was estimated according to Hayes and Velick [8] , the concentration of enzyme active sites was determined by the fluorescent method of Leskovac et al. [9] , and expressed in |xM in the text. Specific activity of enzyme on ethanol was 300 U/mg estimated at pH 9, according to Bergmeyer [1]. All reaction rates were determined in a self-recording double-beam spectrophotometer SPECORD UV VIS, Carl Ziess, Jena (Germany), in thermostated cuvette holders, at 25 °C. All kinetic measurements were performed in 0.1 M sodium phosphate buffer pH 6-8, 0.1 M sodium pyrophosphate pH 9, or 0.1 M sodium glycinate pH 10, supplemented with 0.5 mM EDTA. Extraction of NAD + from the human blood was performed according to Bergmeyer [1]. Human blood was obtained from a voluntary blood donor in the University Hospital Novi Sad, and stabilized in a usual manner. One ml of fresh blood was pipeted directly into 5 ml of cold 0.6 N perchloric acid, and the protein precipitate removed by centrifugation. To 5 ml of the clear supemate, 1 ml of 1M K2HP04 and 0.65 ml of 3N KOH were added to bring pH exactly to 7.0. The solid KC104 was centrifuged and the clear supernate was used as an extract of human blood for the enzymatic determination of NAD + ; the dilution factor human blood/extract was 1:7.75. Results and discussion Chemical and kinetic properties of the enzymatic reaction NDMA is very slowly reduced by NADH in neutral aqueous solutions, with a bimolecular rate constant of 0.4 M_1 s_1. This redox reaction is greatly accelerated in the presence of yeast alcohol dehydrogenase [7]. In aqueous buffers, from pH 6-10, NDMA is enzymatically reduced by NADH into two types of stable reaction products: /f-amino-iV.A-dimethylaniline and Abbreviations-. NDMA, p-nitroso-A( N-dimethylaniline; AD MA, p-ammo-N,N- dimethylaniline; DPD, A(iV-dimethyl-p-phenyl-enediamine Acta Physiologica Hungarica 83, 1995

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