Affinity precipitation of concanavalin A--studies of some underlying mechanisms using dynamic laser light scattering.

Abstract

The final outcome of an affinity precipitation process will depend upon the efficiency of each individual stage involved: the formation of initial affinity complexes, the build-up of a precipitate and the elution of the target protein. Investigations on the first stage were done in this study utilizing a model system. The target protein was the lectin concanavalin A (Con A). Eudragit S-100, a reversibly soluble/insoluble polymer consisting of methyl methacrylate and methacrylic acid, to which the affinity ligand p-aminophenyl-alpha-D-glucopyranoside was coupled, served as the bifunctional ligand (ligand-Eudragit). Owing to the tetrameric structure of Con A, where each subunit has the ability to bind one sugar moiety, and to the multivalency of ligand-Eudragit, a network was formed between the Con A and ligand-Eudragit. It was possible to detect the initial soluble complexes formed by dynamic laser light scattering (DLLS) long before any precipitate could be analysed by transmittance measurements. The rate of complex formation was highly dependent on the ratio between lectin and ligand-Eudragit. It was further shown that the system did not reach equilibrium within the 110 min studied. When the complex formation was studied in the presence of glucose, the build-up rate was decreased to different degrees depending on the sugar concentration used. At high glucose concentrations the complex formation was completely inhibited.