Reversible dissociation of cortisol-transcortin complex by sodium para-chloromercuribenzoate.
Mercurials are considered as sulphydryl group specific reagents and one of them, sodium para-chloromercuribenzoate (PCMB), is currently used for SH titration. It has been shown that cellular steroid receptors are reversibly inactivated by mercurials even when the binding site is occupied by the steroid (Coty, W.A. (1980) J. Biol. Chem. 255, 8035-8037). This is a striking difference with alkylating SH reagents such as iodoacetic acid or N-ethylmaleimide, since these reagents inactivate only steroid-free receptors. In order to explain this discrepancy, we tested, in the present study, the specificity of PCMB on a blood plasma steroid binding protein: human transcortin. This protein presents the advantage, over cellular receptors, of being well characterized and to be available in a pure state. The transcortin-cortisol complex was also reversibly inactivated by PCMB when the reaction was carried out at a high excess of reagent over protein; such conditions are those previously used with steroid receptors. The reversibility was obtained not only with a reducing agent (dithiothreitol) but also with EDTA, which suggests a poor stability of the protein mercurial bond and therefore a nonspecific action. The decrease of activity was the result of a loss of binding sites and Scatchard plot analysis did not reveal any detectable decrease of the affinity constant for cortisol. Transcortin possesses two SH groups per molecule, one of these being buried in native conformation. After blockage of the accessible SH group by aminoethylation, transcortin kept the same activity, but when this aminoethylated transcortin was incubated with PCMB a loss of activity was obtained, although the residual buried SH group was again titrable with Ellman's reagent. Therefore, we can conclude that the action of PCMB on proteins must be interpreted with precaution, since it can induce an inactivation that is SH-independent.
D002729:Chloromercuribenzoates / Q000494:pharmacology*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D006801:Humans; D006854:Hydrocortisone / Q000097:blood*; D007700:Kinetics; D011485:Protein Binding; D013438:Sulfhydryl Compounds / Q000032:analysis; D014156:Transcortin / Q000378:metabolism*; D020245:p-Chloromercuribenzoic Acid
D002729:Chloromercuribenzoates; D013438:Sulfhydryl Compounds; D020245:p-Chloromercuribenzoic Acid; D014156:Transcortin; D004228:Dithionitrobenzoic Acid; D004492:Edetic Acid; D004229:Dithiothreitol; D006854:Hydrocortisone