PROTEOLYSES OF A FLUOROGENIC INSULIN DERIVATIVE AND NATIVE INSULIN INREVERSED MICELLES MONITORED BY FLUORESCENCE EMISSION, REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY, AND CAPILLARY ZONE ELECTROPHORESIS

The preparation and substrate properties of the fluorogenic insulin de rivative N-alpha A1-aminobenzoyl-N epsilon(B29)-Tyr(NO2)-insulin are d escribed. This semisynthetic protein intramolecularly quenched by long -range resonance energy transfer between the donor/acceptor pair 2-ami nobenzoic acid and 3-nitrotyrosine was used to prove the activity of s erine proteases toward substrates of high molecular weight after incor poration in reversed micelles. The proteases investigated, trypsin and alpha-chymotrypsin, were shown to be hydrolytically active in reverse d micellar solvent systems stabilized by cetyltrimethylammonium bromid e or sodium-1,2-bis(2-ethylhexylcarbonyl)-1-ethane sulfonate. Apart fr om fluorometric enzyme assays, methods for monitoring proteolyses in r eversed micelles were elaborated using either reversed-phase high-perf ormance liquid chromatography or capillary zone electrophoresis. Enzym atic digestions of native insulin by the specific protease trypsin and the less specific protease alpha-chymotrypsin were performed. In cont rast to aqueous solution, high but still variable specificity of alpha -chymotrypsin which was dependent on the micellar environment was obse rved. The results promise further insight into the influence of interf acial environments on enzyme action and a novel approach to enzyme-med iated protein modifications by the use of microstructured solvent syst ems. (C) 1994 Academic Press, Inc.