Superoxide and hydrogen peroxide formation during enzymatic oxidation of DOPA by phenoloxidase

Abstract

Generation of superoxide anion and hydrogen peroxide during enzymatic oxidation of 3-(3,4-dihydroxyphenyl)-dl-alanine (DOPA) has been studied. The ability of DOPA to react with has been revealed. EPR spectrum of DOPA-semiquinone formed upon oxidation of DOPA by was observed using spin stabilization technique of ortho-semiquinones by Zn²⁺ ions. Simultaneously, the oxidation of DOPA by was found to produce hydrogen peroxide (H₂O₂). The analysis of H₂O₂ formation upon oxidation of DOPA by using 1-hydroxy-3-carboxy-pyrrolidine (CP-H), and SOD as competitive reagents for superoxide provides consistent values of the rate constant for the reaction between DOPA and being equal to (3.4±0.6)×10⁵ M⁻¹ s⁻¹.

The formation of H₂O₂ during enzymatic oxidation of DOPA by phenoloxidase (PO) has been shown. The H₂O₂ production was found to be SOD-sensitive. The inhibition of H₂O₂ production by SOD was about 25% indicating that H₂O₂ is produced both from superoxide anion and via two-electron reduction of oxygen at the enzyme. The attempts to detect superoxide production during enzymatic oxidation of DOPA using a number of spin traps failed apparently due to high value of the rate constant for DOPA interaction with

• DOPA
• phenoloxidase
• superoxide
• spin trapping
• DOPA-semiquinone
• hydrogen peroxide