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