Much attention has been focused recently on the detection and physical characterization of individual molecules. Using such methods to study the chemical properties, such as reactivity, of single molecules offers the potential to investigate how these might vary from molecule to molecule, and for individual molecules as a function of time. The complex structures of biomolecules such as enzymes make them particularly attractive targets for studying how subtle changes or differences at the molecular level might influence chemical reactivity. We have shown previously that very small (zeptomole) amounts of enzymes can be studied using a fluorescence microassay; single enzyme molecules have also been detected in oil-dispersed droplets by fluorescence microscopy. Here we report the observation of reactions of individual molecules of lactate dehydrogenase (LDH-1), which produces NADH from lactate and nicotinamide adenine dinucleotide (NAD+). When they are present at very low concentrations in a narrow capillary, each enzyme molecule produces a discrete zone of NADH; these can be manipulated electrophoretically and monitored by fluorescence spectroscopy. We find that the activity of individual electrophoretically pure enzyme molecules can vary by up to a factor of four, and that these activities remain unchanged over a two-hour period. We suggest that the origin of the activity differences may lie in the presence of several stable forms of the enzyme.

Continuous monitoring of submillisecond free-solution dynamics of individual rhodamine-6G molecules and 30-base single-stranded DNA tagged with rhodamine was achieved. Fluorescence images were recorded from the same set of isolated molecules excited either through the evanescent field at the quartz-liquid interface or as a thin layer of solution defined by micron-sized wires, giving diffraction-limited resolution of inter-connected attoliter volume elements. The single-molecule diffusion coefficients were smaller and the unimolecular photodecomposition lifetimes were longer for the dye-DNA covalent complex as compared with those of the dye molecule itself. Unlike bulk studies, stochastic behavior was found for individual molecules of each type, and smaller diffusion coefficients were observed.