PROTEASE SUSCEPTIBILITY AND AMINO GROUP ACCESSIBILITY TO TRINITROBENZENESULFONIC ACID OF LEGUMIN DURING ITS GLYCOSYLATION

In vitro digestibility by trypsin and alpha-chymotrypsin of neoglycolegumins, prepared by reductive alkylation of amino groups of pea legumin, was measured for various degrees of glycosylation. With a low extent of glycosylation, the initial rates of hydrolysis by alpha-chymotrypsin were dramatically increased compared to native legumin. When trypsin was used, no increase of the initial rates occurred, because the hydrolysis of new specific cleavage sites, which may have been exposed by a slight expansion of the protein structure, was counterbalanced by a great decrease of rates of hydrolysis of the glycosylated-lysyl peptide bonds. For high glycosylation degrees, the rates of hydrolysis decreased compared to native legumin with both trypsin and alpha-chymotrypsin. Steric hindrance increased as the size of carbohydrate and the degree of glycosylation increased, which predominated over structure expansion in limiting hydrolysis. In native legumin, about 50% of amino groups are accessible to trinitrobenzenesulfonic acid. A progressive decrease of the inaccessible amino groups occurred during glycosylation. Since the inner part of the neoglycoprotein keeps its close packed structure, our results suggest that amino groups are located in the relatively outer part of the molecule.