Electrophoresis is one of the most widely-used techniques in molecular biology, however a thorough and correct theoretical treatment of electrophoresis has proven elusive. This study, in collaboration with Dr. Stuart Allison at Georgia State University, combines experimental measurements with recently developed boundary-element modeling methods aiming at the development of a theory applicable to small polyions in aqueous solvents. DNA oligonucleotides of defined size, charge and charge density will be examined in a range of solvents. Electrophoretic mobility and effective charge will be determined by membrane-confined analytical electrophoresis. Analytical ultracentrifugation will be used to determine solution mass, as well as sedimentation, frictional and viral coefficients. The effective charge will be measured over a wide range of DNA lengths in solvents with varying ionic strengths, salt types and salt valences. Structure-charge relationships also will be explored using oligonucleotides that have formal charge densities that are selectively altered by methylphosphonate substitutions. This allows the examination of the effective charge and hydrodynamics of DNA having the same formal charge but different distributions of charge density. Finally, a ribosomal RNA fragment of defined structure will be examined in order to characterize putative divalent metal ion binding sites. To complement the nucleic acid work, the mobility and effective charge of bovine ribonuclease will be determined over a range of pH and salt concentrations. Structural data indicate that ribonuclease may have a histidine-dependent anion binding site. The modeling of P.I.'s preliminary mobility data indicate that the anion is loosely bound. Experiments will determine the dissociation constant for the ion, define the role of the histidine in anion binding and determine the effects of reversible ion binding on electrophoretic mobility. Taken together, the combination of experimental data and theoretical analysis will allow the development of a broadly applicable theory for electrophoresis. Furthermore, data from this study will define the experimental relationship between the effective charge measured by steady state electrophoresis and other measures of molecular charge. Overall, this work will lay the foundation for the routine determination and manipulation of the effective charge in biochemistry.
Electrophoresis is amongst the most widely used methods in biochemistry, molecular biology and molecular genetics. For example, the technique is at the foundation of the methods used in sequencing DNA for the human genome project. Despite its wide-spread use and fundamental importance, a comprehensive theory for electrophoresis has eluded scientists for the last century. This study is to experimentally analyze models developed in a collaborating laboratory in order to refine the understanding of electrophoresis. It will lay the foundation for the rational manipulation of charge on biomolecules so that better pharmaceutical, agricultural and polymer agents can be developed.
