DIFFERENTIAL-PULSE POLAROGRAPHIC STUDY OF THE REDOX CENTERS IN PEA AMINE OXIDASE

Amine oxidase from pea seedlings was investigated using differential p ulse 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 e nzyme, 50 mmoll(-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 s elected chemical modifications of the active site of the enzyme. The r eaction of pea amine oxidase (PSAO) with diethyldithiocarbamate result ed in the copper-free enzyme and allowed the signal at -1.23 V to be l inked with the reduction of bound copper ions. This peak was also repl aced by the peaks at -0.1 V of the liberated Cu(II) and Cu(I) ions aft er modifying reactions with diethylpyrocarbonate and sodium borohydrid e respectively. The peaks at -0.60 and -0.30 V belong probably to the organic cofactor, as suggested by reactions of the enzyme with carbony l reagents. The parallel decrease in peaks of PSAO was observed in the polarogram of the acting enzyme, in the presence of substrate and com petitive inhibitor. Partial proteolysis of the enzyme by subtilisin at 37 degrees C served to enhance the current of the cofactor reduction. Differential pulse polarograms of hydrolysed pea amine oxidase were c ompared with those of a model compound, quinone, prepared by the air o xidation of topa (2,4,5-trihydroxyphenylalanine). These comparisons co nfirm the quinone nature of the organic cofactor in the active site of PSAO and allow it to be identified with topa quinone.