Figure 4.

pK_a determination. The rRNase (1.25 µM) was reacted with 20 µM of DTNB, CDNB, and NBD-Cl at variable pH values as reported under Materials and Methods. Average pK_a = 7.9 ± 0.1 of the eight reactive cysteines in the rRNase was calculated using (A) DTNB, (B) CDNB, and (C) NBD-Cl (all curves were obtained by fitting to equation (2)). The theoretical curve of an unperturbed protein cysteine (pK_a = 9.1) is reported in the three panels (black line). Data are the average ± SD of three independent experiments. Differences between pK_a values were considered statistically significant by t-test (p < 0.01).