Investigation into the mechanism of λmax shifts and their dependence on pH for the 2-hydrazinopyridine derivatives of two copper amine oxidases

Copper amine oxidases (CuAO), from Escherichia coli (ECAO) and pea seedling (PSAO) were reacted with an excess of the hydrazine derivative 2-hydrazinopyridine (2HP) to form an initial, strongly absorbing adduct, (adduct 1; λmax 420–430 nm) formed by the covalent binding of 2HP with the active site cofactor 2,4,5-trihydroxyphenylalanine quinone (TPQ). Thermal incubation of buffered solutions of adduct 1 (pH 5.65–10.7) or addition of KOH solution (giving a final pH of 13–15) led isosbestically to a dramatic λmax shift yielding adduct 2 (λmax 520–530 nm). For both ECAO and PSAO, an increase in pH resulted in increased formation of adduct 2 with concomitant loss of adduct 1. Maximum adduct 2 formation occurred at pH 9.84 in ECAO and at pH 10.7 in PSAO. Beyond these pH levels, adduct 2 formation occurred to a much lesser extent which was independent of pH, suggesting enzyme denaturation. It is proposed that the conversion of adduct 1 to adduct 2 occurs as a result of hydrazone to azo conversion mediated by loss of a single proton, possibly to the active site base. It is further postulated that adduct formation and subsequent deprotonation can be likened to the substrate and product Schiff base complexes in the reductive half cycle of copper/TPQ containing amine oxidases. As part of this study an extinction coefficient at 280 nm was determined for ECAO by gravimetric analysis. This yielded a value of 2.1×105 M−1 cm−1 giving rise to the need of a correction factor when estimating the protein concentration from an absorbance reading at 280 nm. Using the estimated molecular mass of 160 kDa for the homodimeric ECAO, a correction factor of 0.76 must be applied.