Continuous fluorescence depletion anisotropy (CFDA) measurement of protein rotation

Abstract

Fluorescence depletion anisotropy (FDA) measurements of protein rotation combine the long lifetime of chromophore triplet states with the sensitivity of the fluorescence excitation and detection. Frequency domain (FDA) addresses certain practical limitations of time-domain procedures, such as the need for detector gating, but presents its own difficulties. We have combined time- and frequency-domain FDA methods into an efficient continuous technique (CFDA). Intensity and polarization of a single laser beam are modulated continuously according to a complex, repetitive waveform and fluorescence signals are averaged over recurring waveform periods by a low rearm time signal averager. Methods for extracting triplet decay and absorption anisotropy decay kinetics from data traces generated by arbitrary waveforms have been developed. For a sample of eosin-BSA in 86% glycerol at 9 degree(s)C, rotational correlation times of 77 micrometers and 137 microsecond(s) , initial anisotropies of 0.109 and 0.125 and limiting anisotropies of 0.017 and 0.022, were obtained by CFDA and cuvet FDA, respectively. Differences in results apparently arise from the weighing of decay components in CFDA by triplet lifetimes of individual components.© (2001) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.