Table 1.
Reactivity of rRNase cysteines toward disulfides and thiol reagents. Second-order kinetic constants k (M−1 s−1) for the reaction of cysteines of the rRNase, free Cys, and free GSH with natural disulfides and thiol reagents calculated at pH 7.4 and 25 °C.
| Free Cys | Free GSH | Reduced RNase A | ||||||
|---|---|---|---|---|---|---|---|---|
|
k (M−1 s−1) e |
k (M−1 s−1) e |
k (M−1 s−1) e |
–SH/ mole rRNase |
E.R. f |
k (M−1 s−1) e |
–SH/ mole rRNase |
E.R. f | |
| GSSG | 0.7 ± 0.1 a 0.2 b |
- | 700 ± 40 | 1 | 1000 ± 150 3500 |
9 ± 1 | 5 | 13 ± 2 45 |
| GSSG (8 M Urea) |
0.7 ± 0.1 a 0.2 b |
- | 0.8 ± 0.1 c | 6 | 1.1 ± 0.24 | - | - | - |
| Cystamine | - | 55 ± 2 | 250 ± 80 | 3 | 4.5 ± 1.4 | - | - | - |
| Cystine | - | 12 ± 1 | 53 ± 6 | 6 | 4.4 ± 0.6 | - | - | - |
| Homocystine | - | 0.6 ± 0.1 | 4 ± 1 | 8 | 7 ± 2 | - | - | - |
| DTNB d | - | 20 ± 1 | 2500 ± 100 | 8 | 125 ± 8 | - | - | - |
| CDNB | - | 0.07 ± 0.01 | 0.9 ± 0.1 | 8 | 13 ± 2 | - | - | - |
| NBD-Cl | - | 8 ± 1 | 18 ± 2 | 8 | 2.3 ± 0.4 | - | - | - |
a Experimental value for free Cys (pKa = 8.53) [18]; b theoretical value for unperturbed protein cysteine (pKa = 9.1) [19]; c differences between second-order kinetic constants were not significant; d experimental condition, pH 5.0 and 25 °C; e second-order kinetic constants are the average ± SD from five independent experiments; f enhanced reactivity. The error derived from the propagation of uncertainties (see Materials and Methods).