抄録
Water-soluble carbodiimide [EDC: (l-ethyl-3-(3-dimethylaminopropyl)carbodiimide)] is a useful reagent for chemical modification of carboxyl group of various proteins. Model experiments to establish detailed conditions for the cross-linking reaction with EDC were conducted. Since the reactivity of hexamethylenediamine as a nucleophile was almost comparable to that of glycine ethyl ester, AH-Sephadex and the carboxyl group of aspartylphenylalanine methyl ester were coupled by EDC. From the hydrolyzate of the isolated gel, aspartic acid and phenylalanine methyl ester were identified. When bovine serum albumin (BSA) was incubated with AH-Sephadex and EDC, about 90 % of the BSA was coupled to the gel by 3hr incubation. Moreover, BSA was effectively coupled with the carboxymethyl cellulose (CMC) after activation of the carboxyl groups of CMC with EDC followed by the removal of excess EDC. The latter case would be useful for cross-linking the enzyme molecules to the matrix because of the very mild reaction conditions. For example, endodextranase, which readily lost its activity upon being incubated with EDC (suggesting that a carboxyl group was essential for the enzyme activity), was effectively immobilized to CMC with EDC. This improved reaction step for the cross-linking seemed to be especially useful for the glycosylases, because in most of these enzymes carboxyl groups play a role in the catalytic residue.
