Single Molecule Enzymology of Chymotrypsin using Water-in-oil Emulsion

¹ University of California, Irvine

^* To whom correspondence should be addressed. E-mail: jpbrody{at}uci.edu.

Submitted on October 26, 2004
Revised on November 19, 2004
Accepted on 1 March 2005

	Abstract

Single molecule studies allow us to study subtle activity differences due to local folding in protein, but they are time consuming and difficult because only a few molecules are observed in one experiment. We developed an assay where we can simultaneously measure the activity of hundreds of individual molecules. The assay utilizes a synthetic chymotrypsin substrate that is non-fluorescent before cleavage by chymotrypsin, but is intensely fluorescent after. We encapsulated the enzyme and substrate in micron-sized droplets of water surrounded by silicone oil where each micro-droplet contains less than one enzyme on average. The kinetics is recorded using a microscope and a CCD camera by measuring the fluorescence intensity of the same individual droplet over time. We conclude that enzymatic reactions could occur within this emulsion system, the statistical average activity of individual chymotrypsin molecules is similar to that measured in bulk, and the activity of individual chymotrypsin is heterogeneous.

Key Words: chymotrypsin, enzyme kinetics, fluorescence microscopy, protein dynamics, single molecule enzymology