Differential pulse polarographic study of the redox centres in pea amine oxidase

Abstract

Amine oxidase from pea seedlings was investigated using differential pulse polarography (DPP). Quinone cofactor and copper ions, both bound in the active site, are redox centres of this metalloprotein. In order to keep conditions near to those supporting the active state of the enzyme, 50 mmol1 −1 phosphate buffer, pH 7.0, was used as a medium for measurements. Highly purified amine oxidase was reduced at a dropping mercury electrode (DME) at potentials −1.23, −0.60 V and −0.30 V. The assignment of the electrode processes to the DPP peaks was based on selected chemical modifications of the active site of the enzyme. The reaction of pea amine oxidase (PSAO) with diethyldithiocarbamate resulted in the copper-free enzyme and allowed the signal at −1.23 V to be linked with the reduction of bound copper ions. This peak was also replaced by the peaks at −0.1 V of the liberated Cu(II) and Cu(I) ions after modifying reactions with diethylpyrocarbonate and sodium borohydride respectively. The peaks at −0.60 and −0.30 V belong probably to the organic cofactor, as suggested by reactions of the enzyme with carbonyl reagents. The parallel decrease in peaks of PSAO was observed in the polarogram of the acting enzyme, in the presence of substrate and competitive inhibitor. Partial proteolysis of the enzyme by subtilisin at 37 °C served to enhance the current of the cofactor reduction. Differential pulse polarograms of hydrolysed pea amine oxidase were compared with those of a model compound, quinone, prepared by the air oxidation of topa (2,4,5-trihydroxyphenylalanine). These comparisons confirm the quinone nature of the organic cofactor in the active site of PSAO and allow it to be identified with topa quinone.