Figure 4.

Inactivation of two electron-reduced hTrxR by BCNU. HTrxR (1.6 units/ml; corresponding to 1 nmol subunit/ml) was incubated at 37°C in hTrxR assay buffer (100 mM potassium phosphate/2 mM EDTA, pH 7.4) with various concentrations of BCNU in the presence of 100 μM of NADPH (control without NADPH, closed circles). Aliquots were diluted 100-fold and assayed for residual activity. The apparent half-times of inactivation and BCNU concentrations are: open circles, 1.7 min, 1 mM; closed triangles, 5 min, 500 μM; and closed squares, 17 min, 100 μM. Because the decomposition of BCNU to chloroethyl isocyanate and other reactive species (t_1/2 = 58 min under these conditions) is often rate-limiting (32), the kinetics of EH₂ inactivation can be more complex than suggested by this figure.