Effect of Divalent Cations on the Reduction and Re-formation of the Disulfide Bonds of Deoxyribonuclease
Abstract The availability of stable, purified preparations of bovine pancreatic deoxyribonuclease (mol wt 31,000) has prompted study of the reduction and re-formation of the two disulfide bonds cross-linking its single polypeptide chain. The two disulfide bonds of DNase are quantitatively reduced by mercaptoethanol and similar reagents in minutes at pH 7.2 and room temperature, without need for 8 m urea or any other denaturing agent. The reduced protein is inactive. The unusual ease of reduction of the disulfide bonds is accompanied by remarkable stability of the reduced form of the protein in the absence of divalent cations. After 24 hours of exposure to air oxidation at pH 7.5 in 1 mm EDTA, 95% of the half-cystine residues remain as cysteine. The situation changes if 4 mm Ca++ is added; re-formation of disulfide bonds and full regain of activity occurs in minutes. If Ca++ is present during the reduction, the product contains one disulfide bond and two —SH groups. The molecule is fully active and remains so even after the two —SH groups are carboxymethylated. The same half-reduced, half-oxidized molecule can be obtained by adding Ca++ to the fully reduced protein providing the reducing agent is still present. These experiments, and the requirement for a divalent cation-DNA complex as the substrate for DNase, demonstrate an ion-protein interaction. However, simple gel filtration at neutral pH removes 45Ca++ completely from both the native enzyme and the active form containing only one disulfide bond; thus, the divalent metal is not bound strongly in either case. The primary sequence of DNase is not sufficient to direct a rapid refolding of the reduced protein under conditions which are similar to those that are effective with RNase and a number of other proteins; in order for the information resident in the amino acid sequence of DNase to be fully expressed, and the native three-dimensional structure to be formed, divalent cations are required.