ABSTRACT
Dopaminochrome formation is catalyzed by commercially available purified peroxidases (EC 1.11.1.7) such as horseradish, lacto- and myelo-peroxidase using dopamine, hydrogen peroxide or promethazine sulfoxide as substrates. A rat brain fraction (RBF) catalyzes a similar reaction and its catalytic power increases after preincubation with hydrogen peroxide/ascorbic acid. The activity of both the purified enzymes and the RBF preparation is inhibited by carnosine and characterized by excess substrate inhibition. The enzymes recognize different substrates but show the highest affinity for dopamine. The RBF fraction is strongly buffered against oxidation by compounds such as glutathione and by bioreductive enzymes such as DT-diaphorase (EC 1.6.99.2) which can use as a substrate menadione or dopaminochrome. The rat brain dopamine peroxidizing activity appeared to be mostly bound to the synaptosomal fraction. The reaction catalyzed by the purified peroxidases was followed by electron spin resonance spectroscopy and, unlike that catalyzed by RBF, was shown to produce the signal of a transient dopamine-o-semiquinone radical.
Subject(s)
Brain/metabolism , Dopamine/metabolism , Indolequinones , Indoles/metabolism , Peroxidases/metabolism , Animals , Dopamine/chemistry , Electron Spin Resonance Spectroscopy , Hydrogen-Ion Concentration , Indoles/chemistry , Lactoperoxidase/metabolism , Male , Oxidation-Reduction , Peroxidases/chemistry , Rats , Rats, Wistar , Synaptosomes/metabolismABSTRACT
Lactoperoxidase, when incubated with increasing amounts of promethazine (P) and promethazine sulfoxide (PO) catalyzes the formation of promethazine sulfoxide accompanied by oxygen consumption. An intermediate radical of PO can be detected by electron spin resonance (ESR). Catalase or superoxide dismutase do not inhibit the reaction while dopamine does. The lactoperoxidase-catalyzed formation of dopaminochrome in the presence of hydrogen peroxide is inhibited by P. Both P and PO inhibit acetyl- and butyrylcholinesterase. Purified enzymes were used throughout the study and horseradish peroxidase but not myeloperoxidase had an activity similar to that of lactoperoxidase.
Subject(s)
Peroxidase/metabolism , Promethazine/metabolism , Sulfoxides/metabolism , Catalase/metabolism , Catalysis , Dopamine/metabolism , Horseradish Peroxidase/metabolism , Hydrogen Peroxide/metabolism , Lactoperoxidase/metabolism , Oxidants/metabolism , Oxidation-Reduction , Promethazine/analogs & derivatives , Superoxide Dismutase/metabolismABSTRACT
Promethazine sulfoxide was obtained with a quantitative yield in a horse radish peroxidase-catalyzed reaction of promethazine and hydrogen peroxide and was also prepared by direct chemical synthesis. The enzymatic sulfoxidation of promethazine was studied in vitro as a function of pH, promethazine, and hydrogen peroxide concentration. Promethazine sulfoxide inhibits with an apparent K(i) of 59.7 microM at pH 5.5 the enzymatic reaction, followed spectrophotometrically, polarographically, potentiometrically, and luminometrically. The reaction was also inhibited by ascorbic acid (K(i) 26.8 microM) and glutathione (K(i) 41.8 microM). The spectrophotometric techniques employed, together with ESR spectrometry, allowed the identification of at least three radical species formed in the course of the reaction. Promethazine sulfoxide is devoid of the antioxidant effect exhibited by promethazine on rat brain synaptosomes. The sulfoxide also lacks photosensitizing action, while retaining the neuroleptic effect of the parent compound.
