Abstract
1-Methyl-4-phenyl-2,3-dihydropyridinium (MPDP+), a metabolic product of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), has been shown to generate superoxide radicals during its autoxidation process. The generation of superoxide radicals was detected as a 5,5-dimethyl-1-pyrroline-N-oxide (DMPO).O2- spin adduct by spin trapping in combination with EPR techniques. The rate of formation of spin adduct was dependent not only on the concentrations of MPDP+ and oxygen but also on the pH of the system. Superoxide dismutase inhibited the spin adduct formation in a dose-dependent manner. The ability of DMPO to trap superoxide radicals, generated during the autoxidation of MPDP+, and of superoxide dismutase to effectively compete with this reaction for the available O2-, has been used as a convenient competition reaction to quantitatively determine various kinetic parameters. Thus, using this technique the rate constant for scavenging of superoxide radical by superoxide dismutase was found to be 7.56 x 10(9) M-1 s-1. The maximum rate of superoxide generation at a fixed spin trap concentration using different amounts of MPDP+ was found to be 4.48 x 10(-10) M s-1. The rate constant (K1) for MPDP+ making superoxide radical was found to be 3.97 x 10(-6) s-1. The secondary order rate constant (KDMPO) for DMPO-trapping superoxide radicals was found to be 10.2 M-1 s-1. The lifetime of superoxide radical at pH 10.0 was calculated to be 1.25 s. These values are in close agreement to the published values obtained using different experimental techniques. These results indicate that superoxide radicals are produced during spontaneous oxidation of MPDP+ and that EPR spin trapping can be used to determine the rate constants and lifetime of free radicals generated in aqueous solutions. It appears likely that the nigrostriatal toxicity of MPTP/MPDP+ leading to Parkinson's disease may largely be due to the reactivity of these radicals.
Highlights
electron paramagnetic resonance (EPR) Kinetic Studies of Superoxide Radicals Generated during the Autoxidation of l-Methyl-4-phenyl-2,3-dihydropyridiniuma, Bioactivated Intermediate of Parkinsonian-inducing Neurotoxin l-Methyl-4-phenyl-l,2,3,6-tetrahydropyridine*
Superoxide radicals wasdetected as a 5,5-dimethyl-1- B), which removes the two electrons from the parent tertiary pyrroline-N-oxide (DMPO)*O; spin adduct by spin amine MPTP toform MPDP' [5,6]
These values are in close agree- Such oxidation islikely to involve two successive single elecment to the published values obtained using different tron transfer stepswith the generationof free radical species experimental techniques. These results indicate that superoxide radicals are produced during spontaneous oxidation of MPDP+ and that EPR spin trapping can be used to determine therate constants and lifetimeof free radicals generatedin aqueous solutions
Summary
MPDP+, MPP+, and MPTP were purchased from Research Biochemicals, Inc. (Natick, MA). The spin trap, 5,5-dimethyl-l-pyrroline-N-oxide (DMPO) was purchased from Aldrich. Additional absorption maximum between 200 to 227 nm and which give EPR signals similar to the spin adducts of carbon-centered radicals, nitroxide species (without P-hydrogen splitting) and hydroxyl radical ('OH) [15, 16], it was purified by eluting through an activated charcoal column. Thecommercial DMPO (8.7 M)was diluted to -1.0 M with nitrogen-bubbled triple-distilled water containing 1.0 mM DTPA and passed with vacuum through a prewashed activated charcoal column (5 X 1 cm) under a nitrogen atmosphere. The purified DMPO solution gave no free radical signal seen by EPR and had only one absorption peak in the UV region of the spectrum at 227 nm in aqueous systems with an extinction coefficient of8.0 X lo M" cm" as reported earlier [17]. The. reactions were started by adding the given amount of MPDP+ stock which was prepared in triple-distilled water containing 1.0 mM DTPA. The EPRparameters were set at100 KHz, X band microwave frequency, 9.78 GHz; microwave power, 20 mW (milliwatts); modulation amplitude, 0.71 G (gauss); time constant, 0.64 s; scan time, 500 s; and receiver gain, 1 X lo
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