Single-molecule studies allow the study of subtle activity differences due to local folding in proteins, but 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 nonfluorescent before cleavage by chymotrypsin, but is intensely fluorescent afterward. We encapsulated the enzyme and substrate in micron-sized droplets of water surrounded by silicone oil where each microdroplet contains <1 enzyme on average. A microscope and charge-coupled device camera are used to measure the fluorescence intensity of the same individual droplet over time. Based on these measurements, 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.
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