Shin-ichi OZAKI, Yuji INADA (Toin Univ. Yokohama) and Yoshihito WATANABE
[Keio J. Med. 45, S68 (1996)]
Horseradish peroxidase (HRP) catalyzes the oxidation of a variety of aromatic substrates utilizing hydrogen peroxide, and it is found to be catalytically active in not only aqueous but also nonaqueous media. However, the reaction intermediates in organic solvents have not been clearly defined. Thus, we report here the UV-visible spectra of compound I and II for HRP dissolved in benzene by rapid scanning spectroscopy. HRP is solubilized in benzene homogeneously by the covalent modification of lysine residues with polyethylene glycol. The addition of a stoichiometric quantity of hydrogen peroxide to polyethylene glycolated horseradish peroxidase (PEG-HRP) in benzene gave the compound I chromophore. In the presence of a large excess of guaiacol, compound I is first reduced to compound II, then to the ferric enzyme. Thus, catalytic intermediates in benzene are the ferryl species as established in aqueous buffer. The slower compound I reduction with guaiacol in benzene than in buffer suggests that the accessibility of the active site for the substrates decreases in benzene.