STUDIES ON THE CARBOHYDRATE-BINDING SITES OF THE HEMOLYTIC LECTIN CEL-III ISOLATED FROM THE MARINE INVERTEBRATE CUCUMARIA-ECHINATA

The binding of carbohydrates to the hemolytic lectin GEL-III isolated from the marine invertebrate Cucumaria echinata was studied. Equilibri um dialysis data suggest that GEL-III has two carbohydrate-binding sit es with equal affinity. The binding of specific carbohydrates to GEL-I II induces a decrease in the fluorescence intensity at 339 nm and the shift of the fluorescence emission maximum to a wavelength shorter by 3 nm, owing to the change in the environment of tryptophan. By analyzi ng the change in the fluorescence intensity at 339 nm as a function of the concentration of carbohydrates, the association constants for bin ding of individual carbohydrates to GEL-III were calculated. The resul ts indicate that GaINAc, lactulose, and lactose are bound by GEL-III w ith fairly high affinity among the carbohydrates tested. The pH-depend ence profile of the association constant of lactose suggests that GEL- III binds carbohydrates with highest affinity around pH 5.0. Modificat ion of GEL-III with N-bromosuccinimide produces an oxidized derivative , in which four tryptophan residues/mol were oxidized and had no hemol ytic activity. However, two out of these four tryptophans escaped from the modification in the presence of specific saccharides and the resu lting derivative retained fairly high hemolytic activity.