Fig. 4.
Monitoring disulfide reductase activity through insulin reduction. Comparison of rates of insulin reduction as visualized by light scattering at 600 nm. Unless otherwise noted, 2.5 μM protein was added to assay solution containing 172 μM insulin. The reaction was initiated by addition of 10 μl of 100 mM dithiothreitol, and light scattering was recorded every 60 s until an absorbance of 0.125 was reached. Residual reduction of disulfide bonds is seen in the negative control (dashed line) due to the presence of DTT in the reaction cocktail. (A) Concentration dependence of reductase activity displayed by TrbB. (B) Comparison of reductase activities of TrbB, TrbB C81S/C84S, and DsbCE.coli.