KINETIC EVALUATION OF THE OXIDATION OF PHENOTHIAZINE-DERIVATIVES BY METHEMOGLOBIN AND HORSERADISH-PEROXIDASE IN THE PRESENCE OF HYDROGEN-PEROXIDE - IMPLICATIONS FOR THE REACTION-MECHANISMS

The oxidation of ten 2-substituted 10-(3-(dimethylamino)propyl) phenot hiazines (PHs) by methemoglobin (metHb) and horseradish peroxidase (HR P) in the presence of H2O2 was kinetically analysed based on an enzymi c-chemical second-order reaction with substrate regeneration: PHs are oxidized enzymatically to their radical cations (PH+.) which subsequen tly, in a second order reaction, react further to parent compound and PH-sulfoxide (PHSO). The enzymic reaction rate can be obtained from th e accumulation curves of both radical cation formation and sulfoxide f ormation. In the case of chlorpromazine and promazine both methods gav e similar reaction rates. The rate constant of PH+. decay could also b e determined from the radical cation accumulation curve. This method w as unsuitable for most PHs because they do not yield measurable steady state concentrations of their radicals. The rate constant of reaction of PHs with HRP compound II was also analysed. The logarithm of this rate constant correlated well with the Hammett sigma(para) and the Swa in and Lupton F and R substituent constants, whereas no correlation wi th hydrophobic and steric parameters was found. This indicates that th e interaction of PH with the porphyrin ring, which is the active site of HRP, is predominantly under electronic control. In the case of cata lysis by hemoglobin (Hb), the formation of the reactive Hb form, ferry lHb with a protein radical, appeared to be rate limiting in the oxidat ion of PHs by metHb-H2O2. Differences in the conversion rates of vario us PHs can be explained by a competition! between their electron trans fer reaction to the protein radical and the denaturation reaction(s) i nvolving the protein radical. Our results confirm our earlier observat ion that the mechanism of oxidation by metHb-H2O2 differs from that of the classical peroxidases. In the former case, electron transfer from PH occurs most likely to a tyrosine residue on the globin part, whils t in the latter case electron transfer to the porphyrin moiety takes p lace.