Reactions of pholasin with peroxidases and hypochlorous acid

The ability of myeloperoxidase (MPO) and horseradish peroxidase (HRP) to in duce chemiluminescence (CL) in Pholasin (Knight Scientific, Plymouth, UK), the photoprotein of the Common Piddock Pholas dactylus, was studied. The ox idation of Pholasin by compound I or II of HRP induced an intense light emi ssion, whereas native HRP showed only a small effect. The luminescence obse rved upon incubation of Pholasin with native MPO was diminished by preincub ation with catalase. Considering the high instability of diluted MPO, it is concluded that traces of hydrogen peroxide in water converted MPO to its a ctive forms, compound I and/or II, which are able to oxidize Pholasin. Inde ed, the addition of hydrogen peroxide to a mixture of MPO and Pholasin indu ced an intense burst of light. This emission was enhanced in degree and dur ation in the absence of chloride. Hypochlorous acid, the reaction product o f Cl- and compound I of MPO, was itself able to elicit a luminescent respon se in Pholasin and this luminescence was strongly inhibited by methionine a nd taurine. However, both of these HOCl scavengers only slightly reduced th e light emission induced by MPO/H2O2 in both the presence or absence of chl oride. Thus, hypochlorous acid produced by the MPO/H2O2/Cl- system, under t he conditions described in this study, did not contribute to Pholasin lumin escence. The Pholasin luminescence elicited by formyl-leucyl-methionyl-phen ylalanine (fMLP)-stimulated neutrophils depends both on superoxide anion ra dicals and higher oxidation states of myeloperoxidase (but not on hypochlor ous acid). This is shown by the inhibition of luminescence with superoxide dismutase and potassium cyanide, together with the lack of effect of both m ethionine and taurine. The luminescence response is about eight times great er in cells stimulated with fMLP/cytochalasin B than with fMLP alone. (C) 2 000 Elsevier Science Inc.