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.
The preparation and substrate properties of the fluorogenic insulin derivative N alpha A1-aminobenzoyl-N epilson B29-Tyr(NO2)- 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 alpha-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 alpha-chymotrypsin were performed. In contrast to aqueous solution, high but still variable specificity of alpha-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.
Lenz
V J
VJ
Deutsches Wollforschungsinstitut an der Rheinisch-Westfälischen Technischen Hochschule Aachen, Germany.
Gattner
H G
HG
Leithäuser
M
M
Brandenburg
D
D
Wollmer
A
A
Höcker
H
H
D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000494:pharmacology; D004586:Electrophoresis; D005470:Fluorometry; D007328:Insulin / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D008823:Micelles; D014357:Trypsin / Q000494:pharmacology
D007328:Insulin; D008823:Micelles; D002918:Chymotrypsin; D014357:Trypsin