Quenching of the fluorescence of proteins by silver nitrate

Abstract

The mechanisms by which Ag + may quench protein tryptophanyl fluorescence have been studied. A 1:1 Ag +-tryptophan complex was detected spectrophotometrically and shown to have a k a = 6.5 × 10 3 M −1. The complex was nonfluorescent. Ag + and NO 3 − each caused collisional quenching which proceeded at nearly diffusion-controlled rates in a series of indole-containing compounds. Analysis of the rates by means of Stern-Volmer plots and lifetime measurements showed also that charge and the presence of salt influence the quenching rate constants. The fluorescence of nonsulfhydryl proteins was quenched by AgNO 3 only in concentrations needed for Stern-Volmer quenching of simple indole model compounds. However, the plots for protein quenching were generally nonlinear, a reflection of the heterogeneity of tryptophanyl residues. AgNO 3 quenching increased the polarization of protein fluorescence and decreased the lifetime. Rotational relaxation times were determined from Perrin plots of reciprocal polarization vs fluorescence intensity in the presence of various amounts of AgNO 3. The fluorescence of the sulfhydryl proteins ovalbumin, yeast, and equine liver alcohol dehydrogenases was strongly quenched by AgNO 3 in parallel with the formation of Ag +-mercaptide bonds. The quenching of fluorescence of sulfhydryl proteins was exhibited even in 8 m urea, thus ruling out conformational change as a major basis for the quenching. It was found that Ag + mercaptide bond formation was accompanied by development of an ultraviolet absorption band. The reaction of Ag + with cysteine, for example, could be followed spectrophotometrically. The uv absorption of different silver mercaptides varied with the compound and pH. Since the uv absorption of Ag +-mercaptides extended up to 340 nm, and was also found in Ag +-treated sulfhydryl proteins, energy transfer from excited tryptophans seemed a reasonable basis for the observed fluorescence quenching. This possibility was confirmed by calculation of Förster critical transfer distances for a variety of donor-acceptor (Ag +-mercaptide) pairs. The lifetime of sulfhydryl protein fluorescence was decreased by AgNO 3, but the emission spectrum was relatively little affected, in contrast to previously reported quenching by Hg 2+. Additional mechanisms of fluorescence alteration by Ag + in proteins (e.g., “heavy atom” effect, conformational changes, enhancement of sulfhydryl quenching) are also considered. The spectral effects of Ag + interaction with proteins have the following practical applications:determination of —SH groups; probe of accessibility of binding sites and tryptophan-sulfhydryl distances; determination of rotational relaxation times by Perrin plots of reciprocal polarization vs lifetime; kinetic studies of Ag + interaction with proteins.