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

• Published in: Analytical biochemistry 2005-01, Vol.336 (2), p.243-252
• Main Authors: Moody, Thomas P., Kingsbury, Jonathan S., Durant, Jennifer A., Wilson, Timothy J., Chase, Susan F., Laue, Thomas M.
• Format: Article
• Language: English
• Subjects: Animals; Anions - chemistry; Anions - metabolism; Cattle; Charge of macro-ions in solution; Debye–Hückel–Henry; Electrochemistry; Electrophoresis; Enzyme Stability; Hydrogen-Ion Concentration; Membrane-confined electrophoresis; Ribonuclease, Pancreatic - chemistry; Ribonuclease, Pancreatic - metabolism; Solutions - chemistry; Static Electricity; Steady state electrophoresis; Valence
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2004.09.009

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 p K 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.