Rates of thiol-disulfide interchange reactions involving proteins and kinetic measurements of thiol pKa values

Abstract

Brønsted coefficients have been determined for the rates of thiol-disulfide interchange between low molecular weight thiols and the disulfide groups of four native or modified proteins: DNase (beta nuc congruent to 0.36), lysozyme (beta nuc congruent to 0.55), adenylate kinase(SSCH3)2 (beta nuc congruent to 0.65), and papain(SSCH3) (beta nuc congruent to 0.45). These values are similar to those observed for reductions of oxidized glutathione and Ellman's reagent by a similar set of thiols (beta nuc congruent to 0.5). Glutathione is anomalously slow in reduction of certain protein disulfide groups: although this effect may in part reflect steric hinderance to attack by the relatively large glutathione molecule at disulfides shielded by protein tertiary structure, other (presently undefined) factors appear also to be important, at least in the case of DNase. The rates of reduction of several disulfide derivatives of papain(SSR) by DTT were determined. These data provide estimates of the Brønsted coefficient for the "central" thiol in thiol-disulfide interchange: these estimates fall in the range beta c congruent to -0.25 to -0.43. Rates of reduction of protein disulfide moieties were analyzed by using a Brønsted equation developed previously [Szajewski, R. P., & Whitesides, G. M. (1980) J. Am. Chem. Soc. 102, 2011] to yield pKa values for the participating thiol moieties: in particular, for papain, pKa(Cys-25) = 8.4 at pH 9 and pKa (Cys-25) = 4.1 at pH 6. The thiols of the structurally essential cysteine group of lysozyme seem to have pKa congruent to 11. The advantages and disadvantages of this method for estimating thiol pKa values are discussed.

A Comprehensive Analysis of Selenium-Binding Proteins in the Brain Using Its Reactive Metabolite

“…The reactivity of thiols is closely related to its pK a value. 37) GSH, the most abundant low mass thiol in the cell cytosol, has a pK a of 9.1.…”

Intraspecies Regulation of Ribonucleolytic Activity

“…This decrease could be due to changes in the bRI structure upon RNase 1 binding, as seen upon RNase A binding to porcine RI (29), allowing better access to reactive sulfhydryl groups. Additionally, RNase 1, being more cationic than RNase A (67), could preferentially shift the pK a of nearby cysteine residues (38), thereby favoring the transition from the thiol (RSH) to the more reactive thiolate (RS − ) form (68,69) and facilitating disulfide bond formation (44). hRI, in contrast, has slightly increased oxidative stability upon complex formation with either RNase A or RNase 1 (Table 1).…”

Formation and properties of mixed disulfides between thioredoxin reductase from Escherichia coli and thioredoxin: Evidence that cysteine‐138 functions to initiate dithiol‐disulfide interchange and to accept the reducing equivalent from reduced flavin

“…Reactions of protein thiols with low molecular weight disulfides in a thiol-disulfide interchange reaction are known to correlate with the thiol pK, more than the particular protein tertiary structure (Shaked et al, 1980). The free thiol of C138S, C135S, C32S, and C35S readily forms a TNB-mixed disulfide when reacted with excess DTNB, and the relative rates are given in Table 2.…”