Date of Award

Spring 2003

Project Type

Dissertation

Program or Major

Biochemistry

Degree Name

Doctor of Philosophy

First Advisor

Thomas L Laue

Abstract

Biochemical solutions are composed to a large degree of charged molecules (proteins, DNA, dissolved salts, etc) which can interact and therefore exert a force on each other. Analytical determination of the valence of biological macro-ions contributes to our understanding of the forces involved in both intra- and inter-molecular interactions. This dissertation uses two free solution techniques, membrane confined electrophoresis (MCE) and capillary electrophoresis (CE), to study valence in the context of a series of charge mutants of T4 lysozyme and RNase Sa. The use of these mutants allows attribution of the changes in electrophoretic behavior entirely to changes in charge. Sedimentation velocity and dynamic light scattering were used to verify that the hydrodynamic radius was constant for a given series of charge mutants. Furthermore, this work compares the model predictions of Debye-Huckel-Henry (DHH), Booth, and the more recent boundary element (BE) modeling by Allison with experimental results.

The electrophoretic behavior of a macroion is affected in a complex manner by a variety of forces which 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 hydrodynamic and electrodynamic contributions to the macroion mobility. Membrane confined electrophoresis provides an experimental means by which to gain insight into these contributions. These experiments isolate the effects of charge on electrophoretic mobility and permit a unique test of theories.

Through MCE steady-state measurements, the effective valence (z eff) of both sets of mutants was determined under an ionic strength of 0.11 M at pH 7.5. Further experimentation with T4 lysozyme at low and high ionic strengths was done to investigate how well the models reflected the observed electrodynamic changes. Parallel experiments with capillary electrophoresis were conducted. Diffusion coefficients determined by sedimentation velocity studies were used to convert between capillary electrophoresis and membrane confined electrophoresis results. Although Debye-Huckel-Henry and Booth provide reasonable first approximations, boundary element modeling by Allison and co-workers, using continuum hydrodynamics based on detailed structural information, provides the most accurate predictions of experimentally observed values.

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