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Proceedings Paper

Biomolecular applications of single-molecule measurements: kinetics and dynamics of a single-enzyme reaction
Author(s): Matt Paige; David P. Fromm; William E. Moerner
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Paper Abstract

In this work we describe preliminary experiments in which we have used ultra-sensitive fluorescence microscopy to observe the dynamics of individual enzyme molecules acting upon a substrate. The enzyme, (beta) -galactosidase from E.coli, is specifically immobilized onto a glass substrate while maintaining its functionality. The immobilized protein degrades a fluorogenic substrate to produce a fluorescent product, whose generation can be observed in real time. Individual copies of (beta) -galactosidase can be observed for many minutes, allowing the measurement of a large number of successive substrate turnover events. A rudimentary analysis of these turnovers using autocorrelation functions is presented, and a strong heterogeneity in reaction rates between different molecules is observed. In addition, the challenges inherent in successful surface immobilization of proteins for single-molecule experiments are discussed.

Paper Details

Date Published: 28 March 2002
PDF: 12 pages
Proc. SPIE 4634, Methods for Ultrasensitive Detection II, (28 March 2002); doi: 10.1117/12.463828
Show Author Affiliations
Matt Paige, Stanford Univ. (United States)
David P. Fromm, Stanford Univ. (United States)
William E. Moerner, Stanford Univ. (United States)

Published in SPIE Proceedings Vol. 4634:
Methods for Ultrasensitive Detection II
Charles W. Wilkerson, Editor(s)

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