Purification and Properties of Semisynthetic Staphylococcal Nuclease-T'

Abstract Semisynthetic staphylococcal nuclease-T', the noncovalent complex of peptide fragments synthetic-(6–47) (the peptide, obtained by solid phase synthesis, corresponding to Residues 6 through 47 of nuclease) and native nuclease-T-(49–149) (containing Residues 49 through 149 of nuclease), was isolated in a highly purified form. This material was compared to native nuclease-T', the complex of native fragments nuclease-T-(6–48) and nuclease-T-(49–149). The semisynthetic enzyme derivative has about 90% of the enzymic activity of the native complex against both natural and synthetic substrates, including yeast RNA, heat-denatured salmon sperm DNA, and deoxythymidine 3'-phosphate-5'-p-nitrophenyl phosphate. The Km at 24.3° for this last substrate is 2.2 x 10-4 m for the semisynthetic complex, as compared to 2.1 x 10-4 m for the native. In addition, semisynthetic nuclease-T' is closely similar to native nuclease-T' in tryptophanyl fluorescence emission spectral characteristics, temperature dependence of enzymic activity, and stabilization against proteolytic digestion by the ligands deoxythymidine-3',5'-diphosphate (an inhibitor) and Ca++ (essential for activity). All of the catalytic and structural properties thus show the essential identity of semisynthetic nuclease-T' with the native enzyme derivative.