Quenching of Red Cell Tryptophan Fluorescence by Mercurial Compounds

Abstract

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 (< 5 s) bimolecular 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 ΔH = 6.5 kcal/mol and ΔS= – 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.

Key Words:

• red blood cells
• tryptophan fluorescence
• mercurial quenching
• band 3