Reduction of 2-Carboxy-4'-dimethylaminoazobenzene by Bacillus Diaphorase

Article abstract of DOI:10.1246/bcsj.62.1123

2-Carboxy-4'-dimethylaminoazobenzene (CDMAB) was reductively cleaved into its two component primary arylamines by Bacillus diaphorase together with NADH (pH 6.1, 25 deg C).The reductive cleavage was found to proceed stoichometrically by a spectrofluorometric assay of fluorescent o-aminobenzoic acid and a fluorescent product formed between N,N-dimethyl-p-phenylenediamine and fluorescam,ine.Parallel lines were obtained in Lineweaver-Burk plots of initial rate vs.CDMAB concentration for the reduction.This experimentally observed kinetic behavior is consistent with a reaction scheme in favor of the Ping Pong mechanism in which CDMAB is reduced by a reduced form of diaphorase.From a kinetic formula, the apparent K_m(CDMAB), K_m(NADH), and V_max values are 2.14*1E-5 mol dm^-3, and 1.8*1E-7 mol dm^-3min^-1 per mg of protein,respectively.