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-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.

Nonequilibrium thermodynamics of membrane-confined electrophoresis.

Membrane-confined electrophoresis (MCE) is an electrophoretic transport method in which macromolecules in solution are confined within a cuvette through which a current flows. Small ions that can permeate the membranes permit current flow. The method is the electrophoretic analog to analytical ultracentrifugation. Systems in the MCE instrument are described by nonequilibrium thermodynamics. This description forms the basis of a program, implemented using finite element methods, that can model transport processes in such systems over an extended time, from arbitrary starting conditions to steady state. Issues relevant to the analysis of systems in which macromolecular species are involved in mass-action associations are discussed. Particular attention is given to steady-state electrophoresis, from which measurements of reduced molecular charge are sought. The relationship of such measurements to valence is discussed.

Use of T4 lysozyme charge mutants to examine electrophoretic models.

The electrophoretic mobility of a macro-ion is affected in a complex manner by a variety of forces that arise from the applied field. Coupling of the macro-ion and small-ion flows gives rise to non-conserved forces that are greater than those expected from ordinary hydrodynamic considerations. It is difficult to separate the steady-state hydrodynamic and electrodynamic contributions to the macro-ion mobility. Membrane-confined electrophoresis (MCE), a free solution technique, provides an experimental means by which to gain insight into these contributions. In this work we used MCE steady-state electrophoresis (SSE) of a series of T4 lysozyme charge mutants to investigate these effects and to examine the existing theoretical descriptions. These experiments isolate the effects of charge on electrophoretic mobility and permit a unique test of theories by Debye-Hückel-Henry, Booth and Allison. Our results show that for wild type (WT) T4, where divergence is expected to be greatest, the predicted results are within 15, 8 and 1%, respectively, of experimental SSE results. Parallel experiments using another free-solution technique, capillary electrophoresis, were in good agreement with MCE results. The theoretical predictions were within 20, 13 and 5% of CE mobilities for WT. Boundary element modeling by Allison and co-workers, using continuum hydrodynamics based on detailed structural information, provides predictions in excellent agreement with experimental results at ionic strengths of 0.11 M.