CHARACTERIZATION OF PROTEASE-CATALYZED HYDROLYSIS OF CYANINE-LABELED ANGIOTENSIN USING CAPILLARY ELECTROPHORESIS WITH LASER-INDUCED FLUORESCENCE DETECTION

The hydrolytic cleavage of a cyanine (Cy3)-labeled angiotensin, cataly zed by various proteases, was studied by capillary electrophoresis (CE ) with laser-induced fluorescence detection (LIF). The end-labeled pep tides and the Cy3 diacid internal standard were separated on a 20-mu m X 27-cm capillary with LIF detection (emission, 580 nm) using a frequ ency-doubled solid-state diode laser emitting at 532 nm or a He-Ne las er emitting at 543 nm. Hydrolysis of the Cy3-labeled angiotensin I, ca talyzed by proteinase K, is a sequential process beginning from the C- terminal of the peptide, instead of from random cleavages. Trypsin cat alyzes a specific cleavage of Cy3-angiotensin I to Cy3-Asp-Arg as anti cipated, Using a combination of endopeptidase and carboxypeptidases, t he remnant of the labeled species was characterized by CE-LIF. The met hod provides a general tool for studying the mechanism of protease-cat alyzed hydrolysis of peptide. (C) 1995 Academic Press, Inc.