901 — Electrode process of glutamate dehydrogenase in differential pulse polarography

The anodic and cathodic responses of sulfhydryl groups in glutamate dehydrogenase (GDH) were studied using the d.p.p. method. The current peaks were detected at the potentials Up,anod and Up,cath of approx. −1.45 V and −1.50 V, respectively, versus s.c.e. in phosphate buffer, pH 7. Both the cathodic and the anodic responses of GDH showed autoinhibiting diffusion-controlled adsorption effects. Both peak potentials Up,anod and Up,cath are shifted positively with increasing pH up to pH 8.5 (Up,anod by 40 mV/pH, Up,cath by 60 mV/pH) and then become pH-independent. The preceding chemical reaction (field-induced homolysis) with pH-dependent rate yields the electroactive form, the thiyl radical, through abstraction of the hydrogen atom from the -SH group. The radical could be reduced, yielding the thiol anion after the uptake of one electron. The two-electron anodic oxidation in d.p.p. involves the product of this reduction process and another thiol anion in an adjacent position. The dissociation rate constant of the thiol group near that undergoing homolysis was estimated from the d.p.p. data (log kd = 2.9 ± 0.2).