Protection Afforded by Maltosyl-β-cyclodextrin Against α-Chymotrypsin-Catalyzed Hydrolysis of a Luteinizing Hormone-Releasing Hormone Agonist, Buserelin Acetate

Abstract

Purpose. The present study addresses how maltosyl-β-cyclodextrin (G₂-β-CyD) impacts upon the α-chymotrypsin-catalyzed hydrolysis of buserelin acetate, an agonist of luteinizing hormone-releasing hormone with emphasis upon the direct effect of G₂-β-CyD on the activity of the protease.

Methods. Kinetic and solubility studies were performed in isotonic phosphate buffer (pH 7.4) at 25°C and 37°C. The interaction of α-chymotrypsin with G₂-β-CyD in the buffer solution was examined by differential scanning calorimetry.

Results. G₂-β-CyD decelerated the α-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 nonproductive encounter between a complex of the substrate with G₂-β-CyD and the protease at relatively low CyD concentrations, while the direct inhibitory effect of G₂-β-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 α-chymotrypsin, simultaneously accompanied by the autolysis. By contrast, a two-state thermal unfolding of α-chymotrypsin was observed in the presence of G₂-β-CyD, suggesting reduced proteolytic activity upon binding to G₂-β-CyD.

Conclusions. These results suggest that G₂-β-CyD at higher concentrations inhibits the proteolytic action of α-chymotrypsin through direct interaction with the protease, as well as through the formation of a non-productive complex with the substrate.