Fluorescence of proteins in aqueous neutral salt solutions. I. Influence of anions

Abstract The effects of anions of neutral salts on the fluorescence emission of six proteins as well as on tryptophan and tyrosine were studied in relation to the structure of proteins. Most anions are good quenchers of tryptophyl and tyrosyl fluorescence, free or in proteins. The results with tryptophan and tyrosine indicate involvement of a collisional quenching mechanism due to agreement with Stern–Volmer law. The deactivation of fluorescence probably occurs because of the transition from singlet state to triplet state. Lehrer's modification of Stern–Volmer law was applied to proteins. The effective quenching constants ([ K Q ]eff) and the fraction of fluorescence available ([ f a ]eff) to the quencher are also calculated. In contrast to its effect on tryptophan, CH 3 COO − quenches tyrosyl fluorescence and ClO 4 − does not. The effects on fluorescence of ribonuclease and free tyrosine are similar and without any changes in emission maximum. The anions are divided into three groups based on the effect they have on tryptophan‐containing proteins. (1) NO 3 − , NO 2 − , Br − , and I − have high [ K Q ]eff values and readily quench tryptophyl fluorescence of proteins causing a shift of emission maximum to a shorter wavelength. This change is due to the specific quenching of “exposed” tryptophan residues which are accessible to quenchers and the observed residual fluorescence is from the “buried” tryptophyls. (2) ClO 4 − and SCN − also quench fluorescence of tryptophan in proteins and have lower ([ K Q ]eff) values. In their presence the fluorescence maximum is shifted to a longer wavelength, which indicates the unfolding of a protein with [( f a )eff] = 1. (3) Cl − , CH 3 COO − , and SO 4  do not have a direct effect on the fluorescence of tryptophan. Besides the “direct” effects, “indirect” effects on fluorophors in protein are also seen, pointing out that the neutral salts can interact in more than one manner with proteins. The effectiveness of anions in quenching fluorescence of proteins follows similar sequences which almost resemble the Hofmeister series, viz., SO 4 = , CH 3 COO − ≃ Cl − < ClO 4 − < SCN − < Br − < I − < NO 3 − < NO 2 − .