Date of Award
Doctor of Philosophy
Equilibrium and rate constants for the binding of concanavalin A (Con A) to several sugars at 25(DEGREES)C were determined chromatographically using either the sugar or the protein immobilized on diol-modified silica. In the "reversed-role" mode, three columns having different Con A dimer coverage were prepared, p-nitrophenyl (alpha)-D-mannopyranoside (PNPM) and 4-methylumbelliferyl (alpha)-D-mannopyranoside (MUM) were the solutes chromatographed, and methyl (alpha)-D-mannopyranoside (MDM) was the inhibitor (I) in the mobile phase. In accordance with theory, 1/(capacity factor) (1/k') versus I plots were linear. Association equilibrium constants for PNPM of 24000 M('-1), for MUM of 45000 M('-1), and for MDM of 8300 M('-1), were obtained;Frontal studies in the reversed-role mode were also done. Near-ideal Langmuir plots were obtained for MDM, but not PNPM. This discrepancy for PNPM was attributed to non-specific adsorption. Frontal equations were modified for this non-specific adsorption and the association equilibrium constants determined were in excellent agreement with the results from the zonal studies;Dissociation rate constants were also determined in the reversed-role studies. Plate height data were corrected for mobile phase and stagnant mobile phase effects, from which the rate constant for the sugar, Con A interaction was determined. Dissociation rate constants of 1.9 and 3.1 sec('-1) were obtained for MUM and PNPM, respectively. These studies, however, indicated a deficiency in chromatographic theory, as a plot of total plate height versus k' did not conform to theory;Determination of equilibrium constants for divalent solutes was studied by "normal-role" experiments. Three columns of varying p-aminophenyl (alpha)-D-mannopyranoside (PAPM) coverage and one glucosamine column were prepared. Con A dimer was chromatographed on these columns in a mobile phase containing various concentrations of MDM. Data were fit to three models: the general; independent, equivalent-site; and high-cooperativity models. The data were fit best by the general model. However, discrepancies in the values for the equilibrium constants were noted in comparing the results for the different PAPM columns. The effect of heterogeneity of the immobilized ligand density on the matrix was discussed.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
David J. Anderson
Anderson, David J., "Determination of equilibrium and rate constants for concanavalin A and various sugars using high-performance affinity chromatography " (1986). Retrospective Theses and Dissertations. 8054.