Reaction of tetrahydrobiopterin with superoxide: EPR-kinetic analysis and characterization of the pteridine radical. Free Radic Biol Med 2001 Oct 15;31(8):975-85
Date
10/12/2001Pubmed ID
11595382DOI
10.1016/s0891-5849(01)00680-3Scopus ID
2-s2.0-0035887885 (requires institutional sign-in at Scopus site) 104 CitationsAbstract
It has been shown that BH(4) 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 BH(4). To examine this possibility we determined the rate constant for the reaction between BH(4) 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 BH(4) 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 BH(4) is not a major reaction in vivo. HPLC product analysis showed that 7,8-BH(2) and pterin are the stable products generated from the reaction. The formation of BH(4) cation radical (BH(4)(*+)) was demonstrated by direct EPR only under acidic conditions. Isotopic substitution experiments demonstrated that the BH(4)(*+) is mainly delocalized on the pyrazine ring of BH(4). In parallel experiments, we investigated the effect of ascorbate on 7,8-BH(2) reduction and eNOS activity. We demonstrated that ascorbate does not reduce 7,8-BH(2) to BH(4), nor does it stimulate nitric oxide release from eNOS incubated with 7,8-BH(2). In conclusion, it is likely that BH(4)-dependent inhibition of superoxide formation from eNOS is the mechanism that better explains the antioxidant effects of BH(4) in the vasculature.
Author List
Vásquez-Vivar J, Whitsett J, Martásek P, Hogg N, Kalyanaraman BAuthors
Neil Hogg PhD Associate Dean, Professor in the Biophysics department at Medical College of WisconsinBalaraman Kalyanaraman PhD Professor in the Biophysics department at Medical College of Wisconsin
Jeannette M. Vasquez-Vivar PhD Professor in the Biophysics department at Medical College of Wisconsin
MESH terms used to index this publication - Major topics in bold
Ascorbic AcidElectron Spin Resonance Spectroscopy
Free Radical Scavengers
Free Radicals
Kinetics
Nitric Oxide Synthase
Nitric Oxide Synthase Type III
Oxidation-Reduction
Pterins
Superoxides