Table 1.
Enhanced reactivity and analytical data for the reaction of several thiol reagents with the four reduced proteins in their molten globule-like structures.
| Thiol Reagent | Thiol | Exp. pH | Reactive Cysteines | pKa | k (M−1s−1) | Total E.R. a | E.R. Due to pKa | E.R. Due to Other Factors |
|---|---|---|---|---|---|---|---|---|
| CDNB | GSH | 7.4 | 9.0 | 0.07 | 1 | |||
| GSH | 5.0 | 9.0 | 0.00028 | 1 | ||||
| rBSA | 7.4 | 7 | 7.8 b | 9.8 b | 140 b | 4 | 35 | |
| rLyz | 7.4 | 4 | 7.1 c | 160 c | 2286 c | 4 | 572 | |
| rRNase | 7.4 | 8 | 7.9 d | 0.9 d | 13 d | 3 | 4.3 | |
| rChTg | 5 | 10 | 8.1 e | 0.19 e | 678 e | 2 | 339 | |
| NBD-Cl | GSH | 7.4 | 9.0 | 8 | 1 | |||
| GSH | 5.0 | 9.0 | 0.032 | 1 | ||||
| rLyz | 7.4 | 4 | 7.1 c | 2900 c | 362.5 c | 3 | 121 | |
| rRNase | 7.4 | 8 | 7.9 d | 18 d | 2.3 d | 3 | 0.8 | |
| rChTg | 5.0 | 10 | 8.1 e | 12.6 e | 394 e | 2 | 197 | |
| DTNB f | GSH | 6.0 | 9.0 | 150 | 1 | |||
| GSH | 5.0 | 9.0 | 20 | 1 | ||||
| rBSA | 6.0 | 7 | 7.8 b | 30,000 b | 200 b | 4.3 | 46 | |
| rLyz | 5.0 | 7 | 6.6 c | 3200 c | 160 c | 3.3 | 48 | |
| rRNase | 5.0 | 8 | 7.9 d | 2500 d | 125 d | 3.5 | 36 | |
| rChTg | 5.0 | 1 | 8.1 e | 25,000 e | 1250 e | 3.5 | 357 | |
| rChTg | 5.0 | 9 | 8.1 e | 2300 e | 115 e | 3.5 | 33 | |
| GSSG | Cys | 7.4 | 9.1 g | 0.2 g | 1 g | |||
| Cys | 5.0 | 9.1 g | 0.0008 g | 1 g | ||||
| rBSA | 7.4 | Cys75 | 6.6 b | 250 b | 1250 b | 2.2 | 568 | |
| rLyz | 7.4 | Cys94 | 6.6 c | 600 c | 3000 c | 2.2 | 1364 | |
| rRNase | 7.4 | Cys95 | 7.9 d | 700 d | 3500 d | 2.9 | 1207 | |
| rChTg | 5.0 | Cys1 | 8.1 e | 1.1 e | 1375 e | 2.6 | 529 | |
| Cystamine | GSH | 7.4 | 9.0 | 55 | 1 | |||
| GSH | 5.0 | 9.0 | 0.22 | 1 | ||||
| rLyz | 7.4 | 4 | 6.6 c | 43 c | 0.8 c | 2.2 | 0.4 | |
| rRNase | 7.4 | 3 | 7.9 d | 250 d | 4.5 d | 2.9 | 1.5 | |
| rChTg | 5.0 | 5 | 8.1 e | 0.05 e | 0.23 e | 2.6 | 0.09 | |
| Cystine | GSH | 7.4 | 9.0 | 12 | 1 | |||
| GSH | 5.0 | 9.0 | 0.048 | 1 | ||||
| rLyz | 7.4 | 1 | 6.6 c | 770 c | 64 c | 2.2 | 29 | |
| rRNase | 7.4 | 6 | 7.9 d | 53 d | 4.4 d | 2.9 | 1.5 | |
| rChTg | 5 | 8 | 8.1 e | 0.56 e | 12 e | 2.6 | 4.6 |
a E.R., enhanced reactivity is the second order kinetic constant for a given reaction, normalized to that of an unperturbed protein cysteine; b from Ref. [14]; c from Ref. [13]; d from Ref. [15]; e from Ref. [16]; f E.R. due to pKa from Ref. [17]; g unperturbed protein cysteine from Ref. [8]. Abbreviations: rBSA, reduced bovine serum albumin; CDNB, 1-chloro-2,4-dinitrobenzene; rChTg, reduced chymotrypsinogen; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); GSH, reduced glutathione; GSSG, oxidized glutathione; rLyz, reduced lysozyme; NBD-Cl, 4-chloro-7-nitrobenzofurazan; rRNase, reduced ribonuclease.