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Proceedings Paper

Spectral properties and biological applications of ELF enzyme substrates that yield fluorescent precipitates at the enzymatic activity sites
Author(s): Zhenjun Diwu; Dieter H. Klaubert; Richard P. Haugland
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Paper Abstract

Although coumarin- and fluorescein-based fluorogenic substrates are widely used in a variety of enzyme assays, they are still ill-adapted for continuous assays in the cases of enzymes that have optimal activity below pH 7.0 because coumarins and fluoresceins generally have the maximum fluorescence above pH 10. Additionally the enzymatic products of coumarin- and fluorescein-based fluorogenic substrates are soluble and readily diffuse away from the site of enzyme activity, and are especially troublesome for in vivo applications. We have now developed a variety of 2-(2'- hydroxyphenyl)-4(3H)-quinazolinone (HPQ)-based enzyme substrates that have overcome these limitations. The water- soluble, colorless, and nonfluorescent substrates generate highly fluorescent and photostable precipitates upon enzymatic action without the addition of the secondary trapping agents. Such fluorogenic precipitating substrates ensure fluorescent histochemical imaging of enzymes with cytologically fine resolution and sharp contrast over cellular autofluorescence and therefore inherently enhance sensitivity and specificity of the targeted analysis.

Paper Details

Date Published: 3 May 1999
PDF: 10 pages
Proc. SPIE 3602, Advances in Fluorescence Sensing Technology IV, (3 May 1999); doi: 10.1117/12.347530
Show Author Affiliations
Zhenjun Diwu, Molecular Probes, Inc. (United States)
Dieter H. Klaubert, Molecular Probes, Inc. (United States)
Richard P. Haugland, Molecular Probes, Inc. (United States)

Published in SPIE Proceedings Vol. 3602:
Advances in Fluorescence Sensing Technology IV
Joseph R. Lakowicz; Steven A. Soper; Richard B. Thompson, Editor(s)

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