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) phenothiazines (PHs) by methemoglobin (metHb) and horseradish peroxidase (HRP) in the presence of H2O2 was kinetically analysed based on an enzymic-chemical second-order reaction with substrate regeneration: PHs are oxidized enzymatically to their radical cations (PH+) which subsequently, in a second order reaction, react further to parent compound and PH-sulfoxide (PHSO). The enzymic reaction rate can be obtained from the accumulation curves of both radical cation formation and sulfoxide formation. In the case of chlorpromazine and promazine both methods gave similar reaction rates. The rate constant of PH+. decay could also be determined from the radical 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 Swain and Lupton F and R substituent constants, whereas no correlation with hydrophobic and steric parameters was found. This indicates that the interaction of PH with the porphyrin ring, which is the active site of HRP, is predominantly under electronic control. In the case of catalysis by hemoglobin (Hb), the formation of the reactive Hb form, ferry1Hb with a protein radical, appeared to be rate limiting in the oxidation of PHs by metHb-H2O2. Differences in the conversion rates of various PHs can be explained by a competition between their electron transfer reaction to the protein radical and the denaturation reaction(s) involving the protein radical. Our results confirm our earlier observation 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, whilst in the latter case electron transfer to the porphyrin moiety takes place.
Kelder
P P
PP
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Utrecht University, The Netherlands.
de Mol
N J
NJ
Fischer
M J
MJ
Janssen
L H
LH
D002412:Cations; D002746:Chlorpromazine / Q000378:metabolism; D005609:Free Radicals; D006735:Horseradish Peroxidase / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007700:Kinetics; D008706:Methemoglobin / Q000378:metabolism*; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010640:Phenothiazines / Q000378:metabolism*; D011395:Promazine / Q000378:metabolism; D013329:Structure-Activity Relationship; D013454:Sulfoxides / Q000378:metabolism
D002412:Cations; D005609:Free Radicals; D010640:Phenothiazines; D013454:Sulfoxides; D008706:Methemoglobin; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D011395:Promazine; D002746:Chlorpromazine