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Applications of Capillary Electrophoresis for Studying Serum Albumin Enantioselection of D,L-Tryptophan Analogs

Stinson, Jelynn A.

Abstract Details

2012, Doctor of Philosophy (PhD), Wright State University, Biomedical Sciences PhD.

The pharmacokinetic difference between drug enantiomers is the impetus for developing analytical techniques to assess enantiomeric purity. Capillary electrophoresis (CE) is an analytical technique that is used for characterizing drug-protein binding. The pitfall to using CE for drug-protein binding studies is protein chiral selectors tend to adsorb onto capillary walls and cause changes in electroosmotic flow that lead to decreased enantioselection and migration time irreproducibility between consecutive injections. The experimental parameters for minimizing the adverse effects of protein adsorption are not clear from the literature.

Rinsing protocols to improve enantioselection and migration time repeatability were developed using the tryptophan-bovine serum albumin system as a model. The enantioselection of bovine serum albumin (BSA) could be improved by: 1) increasing separation voltage; 2) using sample buffer ionic strength at least 3 orders of magnitude less than the separation buffer; 3) limiting the equilibration time with separation buffer; and 4) allowing for protein diffusion. Rinsing the capillary with sodium hydroxide, followed by water improved migration time repeatability RSD from 24.7% to 1.8% (n = 4). Drug-protein binding is contingent upon the three dimensional structure of the binding site, and the presence of other competing drug molecules. Drug-drug displacement is difficult to predict and the effects of protein glycation on binding of drugs is not well defined. To highlight the use of CE for addressing questions of biochemical interest, CE was applied to characterize drug-drug displacement and the effects of protein glycation on the enantioselection of drugs by BSA. The tryptophan-bovine serum albumin, 5-fluoro-tryptophan-bovine serum albumin, and 5-hydroxy-tryptophan-bovine serum albumin systems were used as models.

A CE method for studying competitive binding was established using ibuprofen as the displacer molecule. Accurate calculation of selectivity was found to depend on the precomplexation of ibuprofen and BSA. A CE method for studying the effects of protein glycation was developed using BSA containing different degrees of glycation as chiral selectors. The enantioselection of tryptophan analogs by BSA was altered by glycation as reported in other analytical methods. These studies can serve as guidelines for optimizing serum albumin enantioselection and extending its use in other biopharmaceutical applications.

Roger Gilpin, PhD (Advisor)
Gerald Alter, PhD (Committee Member)
Kenneth Turnbull, PhD (Committee Member)
Daniel Ketcha, PhD (Committee Member)
Richard Sherwood, PhD (Committee Member)
191 p.

Recommended Citations

Citations

  • Stinson, J. A. (2012). Applications of Capillary Electrophoresis for Studying Serum Albumin Enantioselection of D,L-Tryptophan Analogs [Doctoral dissertation, Wright State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=wright1346894633

    APA Style (7th edition)

  • Stinson, Jelynn. Applications of Capillary Electrophoresis for Studying Serum Albumin Enantioselection of D,L-Tryptophan Analogs. 2012. Wright State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=wright1346894633.

    MLA Style (8th edition)

  • Stinson, Jelynn. "Applications of Capillary Electrophoresis for Studying Serum Albumin Enantioselection of D,L-Tryptophan Analogs." Doctoral dissertation, Wright State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=wright1346894633

    Chicago Manual of Style (17th edition)