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Volume 271, Number 50, Issue of December 13, 1996 pp. 32168-32173
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Environment-sensitive Labels in Multiplex Fluorescence Analyses of Protein-DNA Complexes

(Received for publication, June 11, 1996, and in revised form, September 30, 1996)

From the Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3206

Fluorescein is widely used for protein labeling because of its high extinction coefficient and fluorescence emission quantum yield. However, its emission is readily quenched by various pathways. We exploit these properties of fluorescein to examine the self-association of a DNA binding protein and determine the amount of the protein in gel-shifted complexes with specific DNA. A construct (HSF_DT^385SH) of the heat shock transcription factor (HSF) was expressed that contains the DNA-binding and trimerization domains, residues 192-385 of HSF, with four additional COOH-terminal residues, GMLC, and then labeled at the COOH-terminal cysteine with fluorescein 5-maleimide to form HSF_DT^385-Fl. The fluorescence increase accompanying the formation of heterotrimers on titration of HSF_DT^385-Fl with HSF_DT^385SH) led to an estimate of 3 × 10¹⁶ M² for the equilibrium constant for trimerization of HSF_DT^385SH. HSF_DT^385-Fl fluorescence also increased 1.7-fold on binding to specific DNA, but not to nonspecific DNA. The protein and DNA content of the several gel-shifted complexes of HSF_DT^385-Fl (_max^em 532 nm) with specific DNA labeled noncovalently with the energy transfer heterodimer TOTAB (_max^em 658 nm) were accurately determined by a two-color fluorescence emission assay with 488 nm excitation.

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