QUENCHING-RESOLVED EMISSION ANISOTROPY - A STEADY-STATE FLUORESCENCE METHOD TO STUDY PROTEIN DYNAMICS

Fluorescence techniques can be used to obtain information about biolog ical objects in a non-destructive manner. One of these techniques is f luorescence quenching which involves a decrease in the fluorescence em ission of a biological object by externally added quenchers. Quencher molecules produce two kinds of quenching: static and dynamic. Static q uenching occurs due to encounter pair formation between quencher and f luorophore molecules, while dynamic quenching requires bimolecular col lisions. Unless one of the mechanisms can be neglected, steady state q uenching experiments cannot provide information on the contributions o f the two processes. However, time-resolved experiments are sensitive only to the dynamic process, and thus provide selective information ab out the relative motion of the quencher and fluorophore. Since the two quenching events are controlled by different physicochemical paramete rs, it is necessary to resolve them. In this paper, we describe a stea dy state method to resolve the static and dynamic quenching constants (rather than time-resolved techniques). Our method is based on the sim ultaneous determination of the fluorescence intensity and emission ani sotropy data and can be regarded as the further development of quenchi ng-resolved emission anisotropy (QREA). Since the steady state anisotr opy and fluorescence lifetime are inversely related, by determining th e steady state fluorescence anisotropy, changes in the fluorescence li fetime (and hence the dynamic quenching process) can be monitored (if other parameters influencing the anisotropy remain constant). We prese nt a theoretical description of the method, computer simulations testi ng its accuracy and results of model experiments with pyridoxamine-pho sphate-labelled lysozyme and acrylamide. By changing the external visc osity, we obtained data on the theoretical inverse relationship betwee n the dynamic quenching constant and viscosity. The application condit ions are also discussed.