Protection afforded by maltosyl-beta-cyclodextrin against alpha-chymotrypsin-catalyzed hydrolysis of a luteinizing hormone-releasing hormone agonist, buserelin acetate.

The present study addresses how maltosyl-beta-cyclodextrin (G2-beta-CyD) impacts upon the alpha-chymotrypsin-catalyzed hydrolysis of buserelin acetate, an agonist of luteinizing hormone-releasing hormone with emphasis upon the direct effect of G2-beta-CyD on the activity of the protease. Kinetic and solubility studies were performed in isotonic phosphate buffer (pH 7.4) at 25 degrees C and 37 degrees C. The interaction of alpha-chymotrypsin with G2-beta-CyD in the buffer solution was examined by differential scanning calorimetry. G2-beta-CyD decelerated the alpha-chymotrypsin-catalyzed hydrolysis of buserelin acetate to give the 1-3 tripeptide and the 4-9 hexapeptide fragments. This deceleration can be explained solely by a non-productive encounter between a complex of the substrate with G2-beta-CyD and the protease at relatively low CyD concentrations, while the direct inhibitory effect of G2-beta-CyD on the proteolytic activity made a considerable contribution to the overall deceleration of the hydrolysis at higher CyD concentrations. Calorimetric studies indicate the presence of intermediate states in the thermal unfolding of alpha-chymotrypsin, simultaneously accompanied by the autolysis. By contrast, a two-state thermal unfolding of alpha-chymotrypsin was observed in the presence of G2-beta-CyD, suggesting reduced proteolytic activity upon binding to G2-beta-CyD. These results suggest that G2-beta-CyD at higher concentrations inhibits the proteolytic action of alpha-chymotrypsin through direct interaction with the protease, as well as through the formation of a non-productive complex with the substrate.