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Proceedings Paper

Fluorescence study of nile red bound to human serum albumin in buffer, denaturant, and reverse micelles
Author(s): Daniel M. Davis; David J. S. Birch; P. R. Gellert; Rodney S. Kittlety; Ronald M. Swart
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Paper Abstract

Nile red non-covalently binds to Human Serum Albumin in at least two binding sites with distinctly different accessibilities for acrylamide quenching. Here, we report on the fluorescence characteristics of the probe-protein complex in various environments using both steady state and time-resolved single proton counting techniques. In particular, fluorescence depolarization measurements demonstrate that the unfolding of a protein by heat is fundamentally different from that using denaturant, regarding the changes in diffusional rotation of the probe at intermediate stages. We also exploit the fluorescence of the probe- protein complex in AOT reverse micelles, to increase our understanding of the nature of compartmentalized biological molecules. The large Stokes shift of Nile red allows the changes in the environment of the probe-protein complex in reverse micelles of varying waterpool size, to be observed. Moreover, comparison of acrylamide quenching of the tryptophanyl residue and bound Nile red in reverse micelles of varying composition, show that there is an induced stability in the nanosecond motions of the protein in reverse micelles of waterpool diameter 80 angstroms. Both far and near UV circular dichroism show that at this waterpool size, the protein structure is nearest it's native state. This waterpool size is about the same size as the central cavity in the molecular chaperone GroEL, which suggests that compartmentalization of proteins `in vivo' aids the protein folding process by inducing stability in the appropriate conformation as well as preventing protein aggregation.

Paper Details

Date Published: 8 May 1995
PDF: 12 pages
Proc. SPIE 2388, Advances in Fluorescence Sensing Technology II, (8 May 1995); doi: 10.1117/12.208504
Show Author Affiliations
Daniel M. Davis, Univ. of Strathclyde (United Kingdom)
David J. S. Birch, Univ. of Strathclyde (United Kingdom)
P. R. Gellert, Zeneca Pharmaceuticals (United Kingdom)
Rodney S. Kittlety, Zeneca Pharmaceuticals (United Kingdom)
Ronald M. Swart, Zeneca Specialties (United Kingdom)

Published in SPIE Proceedings Vol. 2388:
Advances in Fluorescence Sensing Technology II
Joseph R. Lakowicz, Editor(s)

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