Hydroperoxynaphthalimide derivative-mediated oxidation of heme proteins on photoirradiation: Horseradish peroxidase

Horseradish peroxidase (HRP) was photoirradiated in the presence of organic peroxide (1, hydroperoxynaphthalimide derivative) at around 353 nm sind 0 degrees C. This compound bound to a heme pocket of HRP as shown by its inhibitory effect on catalysis by HRP (K-i = 5.5 x 10(-5) M) and subsequently it formed an intermediate in the same way as H2O2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) suggested cleavage of the peptide chain of HRP on photoirradiation with 1. From CD spectra and SDS-PAGE, it was presumed that the destruction of both secondary structure and heme of the enzyme occurred to some extent upon photoirradiation, which resulted in a decrease in the catalytic activity. The absorption spectra also suggested that the heme group of the enzyme was destroyed, and the fluorescence spectra showed that the Try, residue in the photoirradiated HRP was oxidized to N-formylkynurenine by a hydroxyl radical generated from 1. Energy transfer from the excited naphthalimide moiety or hydrogen abstraction also seemed to make some contribution to the alteration of the heme group.