High-performance liquid chromatography of amino acids, peptides and proteins. CIX. Investigations on the relation between the ligand density of cibacron blue immobilized porous and non-porous sorbents and protein-binding capacities and association constants.

A porous silica of nominal 5 microns particle diameter and 30 nm pore size (Nucleosil 300-5) and a non-porous silica of nominal 1.5 microns particle diameter were activated with 3-mercaptopropyltriethoxysilane (MPTS), followed by the immobilization of the triazine dye, Cibacron Blue F3GA. Various biomimetic dye sorbents with graduated ligand densities between 1 mumol/m2 and 0.01 mumol/m2 were prepared. The capacities and the association constants associated with the binding of lysozyme to these sorbents were determined by frontal analysis experiments [J. Chromatogr., 476 (1989) 205-225]. Due to the ability of the Cibacron Blue F3GA-modified silicas to act as mixed mode coulombic and hydrophobic interaction sorbents and the highly charged nature of the surface structure of lysozyme (pl 11), two mobile phase conditions were examined. In one case a 0.1 M phosphate buffer, pH 7.8, was used as the equilibration and loading buffer, in the second case 1 M sodium chloride-0.1 M phosphate buffer, pH 7.8 was employed as the equilibration and loading buffer to monitor the influence of ionic interactions. The elution was performed in each case with a 2.5 M potassium thiocyanate solution. With the porous silica dye sorbents and 1 M NaCl present in the loading buffer, the highest capacity was achieved when Cibacron Blue F3GA was immobilised to the level of 0.1 mumol/m2. In the case of the non-porous silica dye sorbents, the maximum protein capacity was achieved when 0.5 mumol/m2 dye were immobilised onto the support. Evaluation of the frontal breakthrough curves confirmed that the kinetics of adsorption of lysozyme onto the non-porous sorbent were substantially faster than the adsorption of lysozyme onto the porous sorbent due to the absence of pore diffusion effects in case of the non-porous support. Furthermore, the adsorption of lysozyme on both sorbents was faster when no salt was added to the loading buffer, indicating that there is either conformational or reorientation effects operating during the specific binding of the protein to the dye ligand, or that the interaction is proceeding through the participation of a second class of binding sites. The magnitude of the association constants, Ka, for the lysozyme-Cibacron Blue F3GA systems were found to be dependent on the ligand density of the sorbent. With decreasing ligand density, the protein-ligand interaction became stronger, e.g. Ka values became larger. These results confirm earlier observations on the effect of ligand steric compression on the affinate-ligand association constant, e.g. the protein needs sufficient space to interact with the ligand in an optimum way.(ABSTRACT TRUNCATED AT 400 WORDS)