Biophysical Journal 50: 41-53 (1986)
© 1986 the Biophysical Society

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yoshida, T M Articles by Barisas, B G Search for Related Content PubMed PubMed Citation Articles by Yoshida, T M Articles by Barisas, B G

Protein rotational motion in solution measured by polarized fluorescence depletion.

ABSTRACT

A microscope-based system is described for directly measuring protein rotational motion in viscous environments such as cell membranes by polarized fluorescence depletion (PFD). Proteins labeled with fluorophores having a high quantum yield for triplet formation, such as eosin isothiocyanate (EITC), are examined anaerobically in a fluorescence microscope. An acousto-optic modulator generates a several-microsecond pulse of linearly polarized light which produces an orientationally-asymmetric depletion of ground state fluorescence in the sample. When the sample is then probed with light polarized parallel to the excitation pulse, fluorescence recovers over 0-1,000 microseconds as the sum of two exponentials. One exponential corresponds to triplet decay and the other to the rotational relaxation. An exciting pulse perpendicular to the probe beam is then applied. Fluorescence recovery following this pulse is the difference of the same two exponentials. Equations for fluorescence recovery kinetics to be expected in various experimentally significant cases are derived. Least-squares analysis using these equations then permits the triplet lifetime and rotational correlation time to be determined directly from PFD data. Instrumentation for PFD measurements is discussed that permits photobleaching recovery measurements of lateral diffusion coefficients using the same microscope system. With this apparatus, both rotational and translational diffusion coefficients (Dr, Dt) were measured for EITC-labeled bovine serum albumin in glycerol solutions. Values obtained for Dr and Dt are discussed in light of both the PFD models and the experimental system.

This article has been cited by other articles:

				J. N. Forkey, M. E. Quinlan, and Y. E. Goldman Measurement of Single Macromolecule Orientation by Total Internal Reflection Fluorescence Polarization Microscopy Biophys. J., August 1, 2005; 89(2): 1261 - 1271. [Abstract] [Full Text] [PDF]

				M. G. Bell, R. E. Dale, U. A. van der Heide, and Y. E. Goldman Polarized Fluorescence Depletion Reports Orientation Distribution and Rotational Dynamics of Muscle Cross-Bridges Biophys. J., August 1, 2002; 83(2): 1050 - 1073. [Abstract] [Full Text] [PDF]