Design and Characterization of a Labeling Reagent for Covalent Immobilization of Glutathione-S-Transferase

A labeling reagent against S. japonicum glutathione-S-transferase (sjGST), denoted as Br-I, was designed, prepared and characterized for covalent immobilization of sjGST on magnetic submicron particles (MSP). Br-I had a large hydrophobic moiety for binding to one active site of sjGST, an extended flexible bromoacetylamide moiety for covalent linkage to any of the accessible amino/sulfhydryl groups through nucleophilic substitution. In addition, Br-I had an extended carboxyl group for conjugation with aliphatic primary amines on the MSP, besides a flexible sketch to link those moieties together. Free Br-I was both a substrate/pro-inhibitor and a monovalent irreversible inhibitor of sjGST. There was >75% inactivation of sjGST after half an hour with free Br-I in excess to the sjGST active site, but only sulfhydryl groups far away from the active site were modified when their quantities were comparable. After conjugation to the MSP, Br-I selectively immobilized sjGST in the presence of alkaline phosphatase as a competitor. The treatment of immobilized sjGST with the mixture of free Br-I and GSH reduced unfavorable adsorption of small hydrophobic compounds. Therefore, after conjugation to biomaterials, Br-I showed promise for covalent site-specific immobilization of sjGST-fused targeted proteins.