Preparation and characterization of thionyl chloride-activated succinamidopropyl-glass as a covalent immobilization matrix.

Abstract

The reaction of succinamidopropyl-glass with nonaqueous thionyl chloride followed by washing with water produces an activated surface which reacts with amino and thiol groups under nondenaturing conditions. Subsequent treatment of the covalently immobilized species with dilute hydroxylamine at pH 7 and room temperature releases about 80% of those molecules attached through amino groups and about 50% of those attached through thiol groups. The succinamidopropyl group appears to be a minimum requirement for such surface reactivity. The derivatized glass beads are stable during storage, and immobilization is achieved by simply contacting the surface with a solution of the protein or biochemical species to be attached. Moreover, the activated succinamidopropyl sites can be dispersed within an inert surface of glycerolpropyl sites. Such dispersion improves the subsequent release of immobilized protein and should provide a useful technique for preparation of a wide variety of specific matrices for affinity chromatography. It was possible to obtain, in solution, molecules which had been refolded from a completely denatured state in an immobilized form; however, extensive unfolding of proteins in strong denaturants reduced by roughly 50% the amount of protein which could be subsequently released.