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.

Date Published: 28 March 2002
PDF: 12 pages
Proc. SPIE 4634, Methods for Ultrasensitive Detection II, (28 March 2002); doi: 10.1117/12.463828

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