Effect of TrxR activity and NADPH concentration on disulfide bond reduction
To study the effect of TrxR activity and NADPH concentration on disulfide bond reduction, the four combinations with two TrxR activities and two NADPH concentrations were constituted using the cell lysate of the CHO cell culture of molecule 2 and TrxR from Sigma. Table 2 showed the TrxR activity and the NADPH concentration of the four constitutes. The purified molecule 2 was spiked to the final concentration of 1 g/L in each of them. The spiked constitutes were incubated at room temperature under nitrogen overlay for 12h. The obtained samples were analyzed on reduction occurrence by non-reduced SDS-PAGE. The results were given in Table 2. As seen, the reduction did not occur when the NADPH concentration was at 0.25 µM no matter the TrxR activity was either 300 mU/ml or 1200 mU/ml. However, the reduction did occur when the NADPH concentration was 1 µM even at the TrxR activity of 300 mU/ml. The results suggested the NADPH concentration was better correlated with the reduction occurrence than the TrxR activity. To further evaluate the effect of NADPH concentration on reduction, the cell lysate was diluted with 50 mM HAc-NaAc buffer into three concentrations, >1.5 µM, 1 µM, and 0.5 µM. After spiked with the purified molecule 2 to a final concentration of 1 g/L, the samples were incubated at room temperature for 12 h under nitrogen overlay. The reduction by DTT, the positive control, was prepared by incubating molecule 2 with 1 % (W/V) of DTT in the non-reduced SDS-PAGE loading buffer at 75oC for 10 min. Figure 4 showed the non-reduced SDS-PAGE of the incubated samples. As seen, the intensity of the band at ~70 kD, ~50 kD and ~25 kD increased with the NADPH concentration. The level of the reduction at 0.5 µM of NADPH was much less than those at 1 µM and >1.5 µM.