Spectrophotometric analysis of the protective effect of ascorbate against spontaneous oxidation of tetrahydrobiopterin in aqueous solution: kinetic characteristics and potentiation by catalase of ascorbate action

Tetrahydrobiopterin (BH(4)) is oxidized by O(2) readily in aqueous solutions and physiological concentrations of ascorbate have been shown to inhibit this reaction. In order to gain insight into the mechanism of ascorbate effect, a spectrophotometric analysis was applied for the study of the time course of BH(4) oxidation in the presence of various concentrations of ascorbate and the effect of various temperatures on the apparent second-order rate constant of BH(4) oxidation (k(ox)) in the presence or absence of catalase. In 100 micromol/l concentration, ascorbate alone prolonged the half-life time of 36 micromol/l BH(4) 1.4-fold whereas in the presence of catalase 1.85-fold. In the presence of catalase ascorbate decreased the value of k(ox) to 51 +/- 0.67%, whereas in the absence of it only to 64 +/- 0.77% of control (P < 0.01). The extent of ascorbate effect was not dependent on temperature, at least between 22 and 37 degrees C, either in the presence or absence of catalase. In the absence of catalase the apparent Arrhenius activation energies: 57.02 +/- 0.09 kJ/mol (-ascorbate) and 56.77 +/- 2.21 kJ/mol (+ascorbate) whereas in the presence of catalase: 62.72 +/- 1.37 kJ/mol (-ascorbate) and 59.93 +/- 2.84 kJ/mol (+ascorbate, mean +/- S.E.M., n=3) were obtained. The study shows that catalase potentiates the BH(4)-stabilizing effect of ascorbate. It is concluded that removal of H(2)O(2) generated from BH(4) during oxidation by O(2) prevents a decrease of ascorbate concentration, and in the presence of ascorbate the pacemaker step in the overall reaction is the oxidation of BH(4) and not the reduction of the quinonoid BH(2) back to BH(4) by ascorbate.