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Changes of the absorption spectra of milk XDH and DTT-treated W336A mutant after mixing with NADH under anaerobic conditions. Milk XDH (4.9 μM, DTT-treated) (A) or5.1 μM DTT-treated W336A mutant (B) in0.1 M pyrophosphate buffer (pH 7.5) containing 0.2 mM EDTA was anaerobically mixed with 1/20 volume of NADH (10 mM) in the same buffer, and the absorption spectra were recorded at the times indicated. The initial spectra were taken within 5 sec of the addition of NADH.
