Proteolyses of a Fluorogenic Insulin Derivative and Native Insulin in Reversed Micelles Monitored by Fluorescence Emission, Reversed-Phase High-Performance Liquid Chromatography, and Capillary Zone Electrophoresis

Abstract The preparation and substrate properties of the fluorogenic insulin derivative N αA1 -aminobenzoyl- N ϵB29 -Tyr(NO 2 )-insulin are described. This semisynthetic protein intramolecularly quenched by long-range resonance energy transfer between the donor/acceptor pair 2-aminobenzoic acid and 3-nitrotyrosine was used to prove the activity of serine proteases toward substrates of high molecular weight after incorporation in reversed micelles. The proteases investigated, trypsin and α-chymotrypsin, were shown to be hydrolytically active in reversed micellar solvent systems stabilized by cetyltrimethylammonium bromide or sodium-1,2-bis(2-ethylhexylcarbonyl)-1-ethane sulfonate. Apart from fluorometric enzyme assays, methods for monitoring proteolyses in reversed micelles were elaborated using either reversed-phase high-performance liquid chromatography or capillary zone electrophoresis. Enzymatic digestions of native insulin by the specific protease trypsin and the less specific protease α-chymotrypsin were performed. In contrast to aqueous solution, high but still variable specificity of α-chymotrypsin which was dependent on the micellar environment was observed. The results promise further insight into the influence of interfacial environments on enzyme action and a novel approach to enzyme-mediated protein modifications by the use of microstructured solvent systems.