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


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–Hückel–Henry model, compared with expectations based on pKA 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.


Molecular, Cellular and Biomedical Sciences

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Analytical Biochemistry



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