A double-quenching method for studying protein dynamics: Separation of the fluorescence quenching parameters characteristic of solvent-exposed and solvent-masked fluorophors

: A novel experimental method, suitable for separate analysis of the quenching parameters characteristic of solvent-exposed and solvent-masked fluorophors of macromolecules, is described. The method is based on the modified Stern-Volmer analysis and requires simultaneous application of two kinds of quencher: one that can selectively quench the emission of exposed fluorophors (e.g., ionic quenchers) and another that is nonselective (e.g., oxygen or, in many cases, acrylamide), capable of quenching the fluorescence of both exposed and masked groups. In order to examine the accuracy of the model, a computer simulation was performed. The results showed that the errors are comparable to those arising from the conventional quenching experiments. The method is applicable to phosphorescence quenching as well and is extendable to time-resolved measurements (by replacing fluorescence intensities with lifetimes). The method was applied to resolve the quenching parameters of lysozyme fluorescence by the use of iodide as selective and acrylamide as nonselective quenchers. The determination of the acrylamide quenching constant associated with the internal fluorophor, Trp-108 (Kq = 3.5 M-1), permits specific studies on the dynamics of internal regions of the protein. The quenching constant determined for the more exposed residue Trp-62 (Kq = 1.6 M-1) provides local information about the surface independent of the electrostatic effects observed when an ionic quencher is used.