Sensitive quantitation of endonuclease kinetics

Described here is an assay permitting exclusive quantitation of the kinetics of endonucleolysis by a mixed-function nuclease at substrate DNA concentrations much lower than those necessary with other assay methods. It makes possible determination of Michaelis parameters Km and kcat for endonuclease activity of such enzymes. The sensitivity of the assay method to very low DNA concentrations is obtained through use of 32P-labeled DNA as substrate. Digested DNA is electrophoresed, and computerized analysis of an autoradiogram made from the gel gives the extent of digestion. The analysis produces a profile of weight fraction vs. DNA fragment size for each sample taken from a nuclease-DNA reaction mixture. Each experimental profile is compared to a family of theoretical profiles generated by computer using theory of polymer statistics and assuming random cleavage. Theoretical profiles are found whose shapes most closely match those of the experimental profiles. Associated with each best-fitting theoretical profile is a value of the number of cuts made in the DNA sample. These values, plotted against time, give initial reaction velocities. Initial velocities from experiments at different DNA concentrations have been used to obtain Km and kcat for micrococcal nuclease under specific reaction conditions.