Effect of Maillard induced glycation on protein hydrolysis by lysine/arginine and non-lysine/arginine specific proteases

Enzymatic protein hydrolysis is sensitive to modifications of protein structure, e.g. Maillard reaction. In early stages of the reaction glycation takes place, modifying the protein primary structure. In later stages protein aggregation occurs. The specific effect of glycation on protein hydrolysis was studied using α-lactalbumin glycated with D-glucose at 50 °C (0–10 h). This resulted in proteins with different degrees of glycation (DG = 0–63%) without changes in secondary, tertiary and quaternary structure. These glycated proteins were hydrolyzed by lysine/arginine specific proteases (bovine and porcine trypsin) or by non-lysine/arginine specific proteases (Bacillus licheniformis protease (BLP), α-chymotrypsin and subtilisin A). For bovine and porcine trypsin, the maximal degree of hydrolysis decreased linearly with 65% from untreated to maximal glycated protein (DG = 63%). This means trypsin cannot hydrolyze glycated cleavage sites. BLP and subtilisin A hydrolyses were independent of glycation, while α-chymotrypsin cannot hydrolyze cleavage sites with glycated binding sites. This means for non-lysine/arginine specific proteases, the effect of glycation depends on the enzyme sensitivity towards modifications on binding sites. Since not all cleavage sites are efficiently used by the enzymes, the extent of the effects depends on the enzyme selectivity towards cleavage sites (for trypsin) or cleavage sites near glycation sites (for α-chymotrypsin). Combining the results of all proteases, an equation was derived describing the effect of modification of protein primary structure on the extent of hydrolysis based on the enzyme specificity, selectivity and binding site sensitivity.