Reaction of tetrahydrobiopterin with superoxide:: EPR-kinetic analysis and characterization of the pteridine radical

It has been shown that BH4 ameliorates endothelial dysfunction associated with conditions such as hypertension, cigarette smoking, and diabetes. This effect has been proposed to be due to a superoxide scavenging activity of BH4. To examine this possibility we determined the rate constant for the reaction between BH4, and superoxide using electron paramagnetic resonance (EPR) spin trapping competition experiments with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO). We calculated a rate constant for the reaction between BH4 and superoxide of 3.9 +/- 0.2 X 10(5) M(-1)s(-1) at pH 7.4 and room temperature. This result suggests that superoxide scavenging by BH4 is not a major reaction in vivo. HPLC product analysis showed that 7,8-BH2 and pterin are the stable products generated from the reaction. The formation of BH4 cation radical (BH4.+) was demonstrated by direct EPR only under acidic conditions. Isotopic substitution experiments demonstrated that the BH4.+ is mainly delocalized on the pyrazine ring of BH4. In parallel experiments, we investigated the effect of ascorbate on 7,8-BH2 reduction and eNOS activity. We demonstrated that ascorbate does not reduce 7,8-BH2 to BH4, nor does it stimulate nitric oxide release from eNOS incubated with 7,8-BH2. In conclusion, it is likely that BH4-dependent inhibition of superoxide formation from eNOS is the mechanism that better explains the antioxidant effects of BH4 in the vasculature. (C) 2001 Elsevier Science Inc.