Purification and active-site characterization of equine plasma amine oxidase.
An improved purification scheme for an amine oxidase from equine plasma (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 similar 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-nitrophenylhydrazine and phenylhydrazine indicate two active cofactors per dimer. 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 spectrum of EPAO exhibits a sharp signal at g congruent to 2, attributable to the topa semiquinone. Equine plasma amine oxidase possesses novel in vitro substrate specificity; while other mammalian amine oxidases oxidize norepinephrine only slowly or not at all, EPAO displays significant activity toward this biogenic amine.
Carter
S R
SR
Department of Chemistry and Biochemistry, Montana State University, Bozeman 59717-0340.
McGuirl
M A
MA
Brown
D E
DE
Dooley
D M
DM
D006631:Amine Oxidase (Copper-Containing); D000818:Animals; D001665:Binding Sites; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003300:Copper / Q000032:analysis; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D006736:Horses; D007202:Indicators and Reagents; D007700:Kinetics; D046911:Macromolecular Substances; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D010659:Phenylhydrazines; D013053:Spectrophotometry; D013379:Substrate Specificity
D007202:Indicators and Reagents; D046911:Macromolecular Substances; D010659:Phenylhydrazines; C030299:phenylhydrazine; C005307:4-nitrophenylhydrazine; D003300:Copper; D006631:Amine Oxidase (Copper-Containing); D000587:Oxidoreductases Acting on CH-NH Group Donors