Quenching of red cell tryptophan fluorescence by mercurial compounds.
Intrinsic tryptophan fluorescence in red cell ghost membranes labeled with N-ethylmaleimide (N-EM) is quenched in a dose-dependent manner by the organic mercurial p-chloromercuribenzene sulfonate (p-CMBS). Fluorescence lifetime analysis shows that quenching occurs by a static mechanism. Binding of p-CMBS occurs by a rapid (less than 5 s) biomolecular association (dissociation constant K1 = 1.8 mM) followed by a slower unimolecular transition with forward rate constant k2 = 0.015 s-1 and reverse rate constant k-2 = 0.0054 s-1. Analysis of the temperature dependence of k2 gives delta H = 6.5 kcal/mol and delta S = -21 eu. The mercurial compounds p-chloromercuribenzoic acid, p-aminophenylmercuric acetate, and mercuric chloride quench red cell tryptophan fluorescence by the same mechanism as p-CMBS does; the measured k2 value was the same for each compound, whereas K1 varied. p-CMBS also quenches the tryptophan fluorescence in vesicles reconstituted with purified band 3, the red cell anion exchange protein, in a manner similar to that in ghost membranes. These experiments define a mercurial binding site on band 3 in ghosts treated with N-EM and establish the binding mechanism to this site. The characteristics of this p-CMBS binding site on band 3 differ significantly from those of the p-CMBS binding site involved in red cell water and urea transport inhibition.
D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects; D001834:Body Water / Q000378:metabolism; D002729:Chloromercuribenzoates / Q000378:metabolism / Q000494:pharmacology*; D003864:Depression, Chemical; D004305:Dose-Response Relationship, Drug; D004910:Erythrocyte Membrane / Q000187:drug effects* / Q000378:metabolism; D005033:Ethylmaleimide; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D010662:Phenylmercuric Acetate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D014364:Tryptophan; D014508:Urea / Q000378:metabolism; D020245:p-Chloromercuribenzoic Acid
D001457:Anion Exchange Protein 1, Erythrocyte; D002729:Chloromercuribenzoates; D020245:p-Chloromercuribenzoic Acid; C010024:4-aminophenylmercuriacetate; D014364:Tryptophan; D014508:Urea; D005033:Ethylmaleimide; D010662:Phenylmercuric Acetate