The Preparation and Characterization of an Active Derivative of Bovine Pancreatic Deoxyribonuclease A Formed by Selective Cleavage with α-Chymotrypsin

Abstract Bovine pancreatic deoxyribonuclease A was selectively cleaved by digestion with α-chymotrypsin under carefully controlled conditions. The peptide bond in DNase A between tryptophan residue 178 and serine 179 was quantitatively hydrolyzed in less than 15 min by 0.5% α-chymotrypsin at pH 8.5 and 4° when divalent metal ions were absent. The modified DNase A (DNase A (1–178,179–257)) was composed of two chains of molecular weights approximately 21,000 and 9,000 which were covalently linked by a disulfide bond. Enzymatically split DNase A retained about 70% of the activity of the native enzyme in the absence of calcium ions in the enzyme solution and exhibited nearly full activity when 20 mm CaCl2 was first added to the modified DNase. Enhancement of DNase A (1–178,179–257) activity by divalent calcium ions indicated that a greater functional dependence exists for Ca2+ in the modified DNase A than was observed with the native enzyme. The covalent linkage which connects the NH2- and COOH-terminal portions of DNase A (1–178,179–257) is formed between half-cystine residues 170 and 206. Reduction with mercaptoethanol inactivated both the native and cleaved DNase A. Unlike native DNase A, reduced DNase A (1–178,179–257) could not be reactivated by the addition of Ca2+ in the presence of 50 mm mercaptoethanol. Activity was restored to reduced DNase A (1–178,179–257), however, to the extent of 90% of that of the native enzyme when 20 mm CaCl2 was added to the reduced chains after the excess mercaptoethanol had been removed. DNase A (1–178,179–257) was further digested with a carboxypeptidase from yeast which quantitatively released 5 amino acid residues, all from the newly formed COOH terminus at position 178 in the modified enzyme. DNase A (1–173,179–257), produced by the carboxypeptidase from yeast, was more than 80% as active as native DNase A in the presence of 20 mm CaCl2. The sequence of residues 174 through 178 is known to be -Thr-Ser-Ser-Gln-Trp-, and threonine 174 is only 4 residues removed from the half-cystine residue at position 170 which participates in the essential —S—S— bond of DNase. These results indicate that a number of the amino acid residues located in the central portion of the peptide chain of DNase are structurally and functionally expendable. The fact that DNase A can be readily cleaved without loss of enzyme activity makes the possibility of proteolysis during preparation of the enzyme a very likely complication. Acrylamide gel electrophoresis, in the presence of sodium dodecyl sulfate and mercaptoethanol, of a sample of DNase A prepared by chromatography on phosphocellulose, indicated the presence of two minor bands which corresponded to the chains from DNase A (1–178,179–257). Chromatography of DNase on DEAE-Sephadex and then a re-chromatography of the pooled DNase fraction on phosphocellulose led to a homogeneous DNase A which was stable at pH 8.0.