149 Clinica Chimica zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Acta, 107 (1980) 149-154 0 ElsevierlNorth-Holland Biomedical Press CCA 1503 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA STOICHIOMETRY OF THE NADH-OXIDOREDUCTASE REACTION FOR DEHYDROGENASE DETERMINATIONS STEPHEN BARNES * and JERRY G. SPENNEY ** Division of Gastroenterology, University of Alabama M edical Center and the Veterans Administration Hospital, Birmingham, AL (U.S.A.) (Received January 7th, 1980) Summary The NADH oxidoreductase reaction with resazurin was most rapid at pH 6.5. FMN (10 pmol/l) markedly stimulated the reaction, and the optimal concentra- tion of resazurin was 50 pmol/l. The oxidation of NADH by NADH oxido- reductase with resaruzin as electron acceptor gave a variable yield of fluores- cent product, resorufin. The yield was pH dependent and was greatest at pH 6.5. Measurements of oxygen consumption in the reaction mixture demon- strated that dissolved O2 was an alternative electron acceptor. The increased yield of resorufin at pH 6.5 was due to more rapid reduction of resazurin rather than oxygen. In contrast, at pH 9.0 oxygen was the preferred electron acceptor. The sensitivity of assays utilizing this indicator reaction can be improved by these optimized conditions. Introduction The chromogen resazurin was introduced by Guilbault and Kramer [ 1,2] for quantitative analysis of dehydrogenase enzymes. NADH produced by the dehy- drogenase was subsequently used for reduction of resazurin by NADH oxido- reductase. NADH + H’ + resazurin NADH oxldo- x resorufin + NAD’ + H,O (1) zyxwvut * Present address: Connective Tissue Laboratory. Diabetes Hospital, University of Alabama Medical Cen- ter, Birmingham, AL 35294, U.S.A. ** Correspondence should be addressed to Jerry G. Spenney, Division of Gastroenterology, University of Alabama Medical Center. University Station, Birmingham. AL 35294, U.S.A. Abbreviations: NAD. @-nicotinamide adenine dinucleotide, oxidized form; NADH, P-nicotinamide adenine dinucleotide, reduced form; FMN, flavin mononucleotide; DCPIP, 2,6-dichlorophenol-indophenol.