Use of T4 lysozyme charge mutants to examine electrophoretic models

Abstract

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.