CHARACTERIZATION OF PROTEASE-CATALYZED HYDROLYSIS OF CYANINE-LABELED ANGIOTENSIN USING CAPILLARY ELECTROPHORESIS WITH LASER-INDUCED FLUORESCENCE DETECTION
Fta. Chen, CHARACTERIZATION OF PROTEASE-CATALYZED HYDROLYSIS OF CYANINE-LABELED ANGIOTENSIN USING CAPILLARY ELECTROPHORESIS WITH LASER-INDUCED FLUORESCENCE DETECTION, Analytical biochemistry, 225(2), 1995, pp. 341-345
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.