Valence and anion binding of bovine ribonuclease A between pH 6 and 8

• DOI: 10.1016/j.ab.2004.09.009
• Publication Date: 2004-11-25T17:35:09Z
• Authors: Thomas P. Moody; Jonathan S. Kings...; Jennifer A. Durant; Timothy J. Wilson; Susan F. Chase; Thomas M. Laue
• Keywords: Divalent

Several studies have shown that divalent anion binding to ribonuclease A (RNase A) contributes to RNase A folding and stability. However, there are conflicting reports about whether chloride binds to or stabilizes RNase A. Two broad-zone experimental approaches, membrane-confined electrophoresis and analytical ultracentrifugation, were used to examine the electrostatic and electrohydrodynamic characteristics of aqueous solutions of bovine RNase A in the presence of 100 mM KCl and 10 mM Bis-Tris propane over a pH range of 6.00-8.00. The results of data analysis using a Debye-Huckel-Henry model, compared with expectations based on pK(A) values, are consistent with the binding of two chlorides by RNase A. The decreased protein valence resulting from anion binding contributes 2-3 kJ/mol to protein stabilization. This work demonstrates the utility of first-principle valence determinations to detect protein solution properties that might otherwise remain undetected.