Fig. 5.

Effect of SNP on SH-protected TPH. TPH–CC was prepared and adsorbed to GSH-affinity beads as described and exposed to 50 μm DTNB at 30°C for 15 min without DTT. Controls were incubated with buffer alone. Beads were washed three times with 50 mm Tris-HCl, pH 7.5, at 4°C to remove DTNB. After this initial treatment, control TPH and DTNB-treated TPH were exposed to 100 μm SNP at 4°C for 30 min, and beads were washed free of SNP. The last step involved exposure of each enzyme preparation to 10 mm DTT at 4°C for 30 min. Controls were incubated with buffer alone. After a final three washes to remove DTT, residual TPH activity was assayed without DTT in the presence of 100 μm iron. The results are presented as % control TPH activity (45.2 nmol of 5-HTP · min⁻¹ · mg⁻¹) and are the mean ± SEM of four independent experiments run in duplicate. The effects of DTNB and SNP were statistically significant from controls by Student’s t test; *p < 0.01. The effect of 10 mm DTT was also significantly different from control after these multiple incubation steps (*p < 0.01). The effect of DTT (10 mm) to reverse DTNB inactivation of TPH was significant for both control and SNP-treated enzyme (**p < 0.01 for each by Student’s t test).