Catalytic Properties and Specificity of the Extracellular Nuclease of Staphylococcus aureus

A spectrophotometric assay is described for staphylococcal nuclease, based on the increase in absorbance at 260 mµ which accompanies deoxyribonucleic acid and RNA hydrolysis. Initial velocities are proportional to enzyme concentration over a 70-fold range. The enzyme has greater affinity for DNA than for RNA, and activity is greater with heat-denatured DNA than with native DNA. No inhibitory products accumulate during the reaction. The enzyme is stable at pH values as low as 0.1, and in a concentration of 0.15 mg per ml there is no loss of activity after boiling (20 min). Dilute solutions are protected from heat inactivation by a mixture of albumin and Ca++ as well as by denatured DNA. The optimum pH for RNase and DNase activities is between 9 and 10, depending on the Ca++ concentration. At higher pH values, less Ca++ is required. The inhibitory effect of high Ca++ concentrations is more pronounced at higher pH values. Considerable DNase but no RNase activity results if Ca++ is replaced by Sr++, while Fe++ and Cu++ cause minimal activation. A number of heavy metal cations inhibit DNase and RNase activities competitively with Ca++; Hg++, Zn++, and Cd++ are the most potent of these. Activities resulting from combinations of DNA and RNA with Ca++ or Sr++ suggest that these substrates are hydrolyzed by the same or closely related regions on the enzyme. Enzyme activity toward DNA and RNA is strongly inhibited by 5'-phosphoryl (not by 2'- or 3'-phosphoryl) derivatives of deoxyadenylic, adenylic, and deoxythymidylic acids, and deoxythymidine 3',5'-diphosphate is the most potent inhibitor. High activity is obtained with polyadenylic acid compared to polyuridylic acid, polycytidylic acid, and RNA. These findings are consistent with the known action of the enzyme (cleavage of the 5'-phosphoryl ester bond), and suggest that the differential activity toward DNA and RNA results at least in part from differences in the affinity toward the constituent bases of these nucleic acids.