Enzymatic reduction of 5-phenyl-4-pentenyl-hydroperoxide: detection of peroxidases and identification of peroxidase reducing substrates.

5-Phenyl-4-pentenyl-hydroperoxide (PPHP) is reduced to 5-phenyl-4-pentenyl-alcohol (PPA) by plant and animal peroxidases in the presence of reducing substrates. PPHP and PPA are rapidly isolated with solid phase extraction, separated by isocratic reverse-phase high-performance liquid chromatography, and quantitated with a fixed-wave-length ultraviolet detector. The procedure described is suitable for detecting peroxide-reducing enzymes, determining the kinetic properties of heme- and non-heme-containing peroxidases, and evaluating oxidizable compounds as reducing substrates for peroxidases. Horseradish peroxidase (HRP) and phenol reduce PPHP with a Km for phenol of 252 microM and a turnover number of 1.05 X 10(4) min-1. Under similar conditions, the Km of HRP for PPHP is 18 microM in the oxidation of guaiacol. A series of 21 compounds was evaluated for the ability to serve as reducing substrates for HRP. The results indicate that the procedure described can not only identify compounds that are reducing substrates but also rank them for relative activity. This may provide a new method with which to identify novel antithrombotic, antimetastatic, or anti-inflammatory drugs as well as to detect and characterize mammalian peroxidases.