PURIFICATION AND ACTIVE-SITE CHARACTERIZATION OF EQUINE PLASMA AMINE OXIDASE

An improved purification scheme for an amine oxidase from equine plasm a (EPAO), a nonruminant source, is described and the protein's active- site is characterized. EPAO is dimeric and contains one Type-2. Cu(II) ion per monomer. The EPAO Cu(II) site is spectroscopically very simil ar to the Cu(II) sites in other amine oxidases. Unlike the extensively investigated nonruminant amine oxidase from porcine plasma, EPAO does not display half-of-the-sites reactivity; titrations with p-nitrophen ylhydrazine and phenylhydrazine indicate two active cofactors per dime r. This cofactor is determined to be the same as that of other copper- containing amine oxidases, 6-hydroxydopa quinone (topa quinone). Upon anaerobic reduction with substrate at ambient temperature, the EPR spe ctrum of EPAO exhibits a sharp signal at g = 2, attributable to the to pa semiquinone. Equine plasma amine oxidase possesses novel in vitro s ubstrate specificity; while other mammalian amine oxidases oxidize nor epinephrine only slowly or not at an, EPAO displays significant activi ty toward this biogenic amine.