Single Molecule Reactions of the Enzyme LDH and of Restriction Endonucleases in the Fluorescence Microscope

Abstract

Two types of single molecule enzyme reactions can be directly observed in the fluorescence microscope: reactions, which convert nonfluorescing small substrate molecules into fluorescing products (or vice versa) and reactions of enzymes on macromolecules stained by a fluorescence dye or visualized otherwise. As an example of the first type of reaction, the conversion of nonfluorescent NAD⁺ into fluorescing NADH, or vice versa, by a few molecules of lactate dehydrogenase in femtodroplets is described. The femto-droplet-pipetting method is essentially a subattomol technique with high accuracy. Lineweaver Burk plots are obtained with approximately the kinetic constants of the enzyme known from conventional biochemistry. On the other hand, the femtodroplet-in-substrate method allows the observation of the action of individual enzyme molecules. The second type of single molecule enzyme reactions is the sequence-specific cutting of individual DNA molecules held by optical tweezers. It is shown that such molecules can be characterized by the cutting (restriction) pattern generated by the restriction endonucleases ApaI, SmaI and EcoRI.