Single molecules of alkaline phosphatase are captured in a capillary filled with a fluorogenic substrate. During incubation, each enzyme molecule creates a pool of fluorescent product. After incubation, the product is swept through a high-sensitivity laser-induced fluorescence detector; the area of the peak provides a precise measure of the activity of each molecule. Three studies are performed on captured enzyme molecules. In the first study, replicate incubations are performed on the same molecule at constant temperature; the amount of product increases linearly with incubation time. Single enzyme molecules show a range of activity; the most active molecules have over a 10-fold higher activity than the least active molecules. In the second study, replicate incubations are performed on the same molecule at successively higher temperatures. The activation energy of the reaction catalyzed by a single molecule is determined with high precision. Single enzyme molecules show a range of activation energy; microheterogeneity extends to thermodynamic properties of catalysis. The average activation energy is within experimental error of the activation energy obtained from analysis of a bulk sample. These results are consistent with the first postulate of statistical thermodynamics:  a thermodynamic property obtained from the time average of an individual molecule is identical to that produced by an ensemble average over a large number of molecules. In the third study, the activity of single enzyme molecules is measured after partial heat denaturation. The number of active molecules decreases in proportion to the extent of denaturation. However, the activity of the surviving molecules is experimentally indistinguishable from the activity of control enzyme. Thermal denaturation of alkaline phosphatase is a catastrophic process, wherein the molecule undergoes irreversible conversion to an inactive form.

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Capillary electrophoresis with laser-induced fluorescence detection was used to develop a universal, highly specific protease assay. In this method, a peptide, biotinylated at the N-terminus, is labeled with fluorescein at a lysine residue near the C-terminus. Impurities are removed from the fluorescence labeling mixture by solid-phase extraction of the substrate on immobilized streptavidin, followed by extensive washing. The purified fluorescent substrate is dissociated from the streptavidin and incubated with the protease. The peptide sequence between the biotin and fluorescent label contains the cleavage sequence of the protease of interest. After cleavage, the fluorescent product does not contain a biotin group. A second solid-phase extraction is used to remove unreacted substrate to dramatically lower the background signal. The product is detected by capillary electrophoresis, which provides powerful discrimination against products generated by nonspecific proteases. With chymotrypsin as a test protease, product was detected with as little as 10 pg/mL (4.6 x 10(-13) M) chymotrypsin, or 5 amol of enzyme in the 10-microL sample volume.