Reactivation of horseradish peroxidase with imidazole for continuous determination of hydrogen peroxide using a microflow injection-chemiluminescence detection system.

A method for reactivation of inactivated horseradish peroxidase (HRP) was studied and exploited in an assay for hydrogen peroxide (H(2)O(2)). Addition of imidazole into a mobile phase made continuous determination of hydrogen peroxide (H(2)O(2)) possible by micro fl ow injection based on horseradish-catalysed luminol chemiluminescence. For reproducible determination of H(2)O(2) with HRP, the inactivation of HRP via protonation of the active sites of HRP caused by reaction with H(2)O(2) must be avoided. We successfully reactivated protonated HRP (inactive HRP) with exogenous imidazole in the mobile phase of the micro fl ow injection system. The imidazole successfully removed the attached proton from the inactive sites of the HRP. This assay was reproducible (within-run reproducibility, CV = 4.0%) and the detection limit for H(2)O(2) was 5 pmol.