Reduction of 2-Carboxy-4′-dimethylaminoazobenzene by <i>Bacillus</i> Diaphorase

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  • Reduction of 2-carboxy-4'-dimethylaminoazobenzene by Bacillus diaphorase.

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2-Carboxy-4′-dimethylaminoazobenzene (CDMAB) was reductively cleaved into its two component primary arylamines by Bacillus diaphorase together with NADH (pH 6.1, 25 °C). The reductive cleavage was found to proceed stoichiometrically by a spectrofluorometric assay of fluorescent o-aminobenzoic acid and a fluorescent product formed between N,N-dimethyl-p-phenylenediamine and fluorescamine. 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 Km(CDMAB), Km(NADH), and Vmax values are 2.14×10−5 mol dm−3, 7.35×10−5 mol dm−3, and 1.8×10−7 mol dm−3 min−1 per mg of protein, respectively.

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