An immunological approach to the conformational equilibrium of staphylococcal nuclease.

The conformational equilibrium constant, K_conf, of staphylococcal nuclease, describing the equilibrium between the native conformation and non-native or disordered conformations, has been estimated using an immunologic method and an interpretive model. Using goat antisera prepared toward a conformationally disordered nuclease fragment (99–149), antibodies specific for the disordered form of the helix-rich sequence 99 to 126, anti-(99–126)_R, were isolated by sequential immunoabsorption. Anti-(99–126)_R forms soluble 7 S complexes with fragment (99–149), but this interaction may be inhibited by a large excess of nuclease. By using fragment (99–149) preferentially carbamylated at the α-amino terminus with KN¹⁴CO and rabbit anti-goat immunoglobulin to distinguish between antibody-bound and free fragment (99–149), an assay for the quantitation of the degree of inhibition of anti-(99–126)_R. (99–149) complex formation by nuclease was developed.Using a formal analysis based on the hypothesis that nuclease is in a conformational equilibrium between a folded and unfolded form and that anti-(99–126)_R binds effectively only to the unfolded form, the K_conf of nuclease was estimated to be 2900. In the presence of the ligands Ca(II), or Ca(II) and thymidine-3′,5′-diphosphate, K_conf values of 6500 and 30,000 to 50,000 were estimated, respectively. The K_conf of nuclease at 4 °C and 39 °C was 3900 and 400, respectively.