Quenching-resolved emission anisotropy: a steady state fluorescence method to study protein dynamics

Abstract Fluorescence techniques can be used to obtain information about biological objects in a non-destructive manner. One of these techniques is fluorescence quenching which involves a decrease in the fluorescence emission of a biological object by externally added quenchers. Quencher molecules produce two kinds of quenching: static and dynamic. Static quenching occurs due to encounter pair formation between quencher and fluorophore molecules, while dynamic quenching requires bimolecular collisions. Unless one of the mechanisms can be neglected, steady state quenching experiments cannot provide information on the contributions of the two processes. However, time-resolved experiments are sensitive only to the dynamic process, and thus provide selective information about the relative motion of the quencher and fluorophore. Since the two quenching events are controlled by different physicochemical parameters, it is necessary to resolve them. In this paper, we describe a steady state method to resolve the static and dynamic quenching constants (rather than time-resolved techniques). Our method is based on the simultaneous determination of the fluorescence intensity and emission anisotropy data and can be regarded as the further development of quenching-resolved emission anisotropy (QREA). Since the steady state anisotropy and fluorescence lifetime are inversely related, by determining the steady state fluorescence anisotrophy, changes in the fluorescence lifetime (and hence the dynamic quenching process) can be monitored (if other parameters influencing the anisotropy remain constant). We present a theoretical description of the method, computer simulations testing its accuracy and results of model experiments with pyridoxamine-phosphate-labelled lysozyme and acrylamide. By changing the external viscosity, we obtained data on the theoretical inverse relationship between the dynamic quenching constant and viscosity. The application conditions are also discussed.