QUANTITATIVE ASPECTS OF AFFINITY ADSORPTION (PROTEIN BINDING)

Available from UMI in association with The British Library.; This thesis addresses two aspects of ligand density effects in affinity adsorption.; Theory (Hubble (1987) Biotech. Bioeng. 30 208-215) predicted that small but finite concentrations of free ligand should enhance the binding of a positively cooperative protein to an immobilised ligand. This was investigated experimentally using two model systems. (i) With yeast glyceraldehyde-3-phosphate dehydrogenase binding to immobilised NAD{dollar}sp+{dollar} or AMP Sepharose matrices no evidence for enhanced binding was apparent as NAD{dollar}sp+{dollar} concentration was increased. Using an AMP cellulose matrix, however, an approximately 4 fold enhancement was observed. Possible explanations for the differing behaviour of the cellulose and Sepharose matrices are discussed. (ii) With bovine liver fructose bisphosphatase binding to phosphocellulose matrices, no evidence for enhancement of binding was observed as a function of AMP concentration, using frontal analysis to measure capacity.; The second aspect explored is the effect of immobilised ligand density on protein binding characteristics for ligands immobilised on soluble polymers. The binding of lysozyme to Cibacron Blue 3G-dextran conjugates was used as a model system.; Dye-dextran conjugates with different ligand densities were synthesised and characterised. Methods were developed using gel-permeation chromatography and spectral titration to enable liquid phase protein adsorption isotherms to be measured. Results for lysozyme binding indicated that no steric hindrance even at the highest dye density tested (1 dye residue per 16 glucosyl residues) hence a constant fraction of the immobilised dye bound specifically to lysozyme regardless of dye density. The operational affinity of conjugates for lysozyme increased with increasing dye density. The possible mechanisms for this effect and its implications are discussed.; The effect of buffer composition and ionic strength on lysozyme binding to dye dextran-conjugates was also investigated, and preliminary data is presented to demonstrate the feasibility of using spectral titration for kinetic as well as equilibrium studies of protein binding.