Characterization of Protease-Catalyzed Hydrolysis of Cyanine-Labeled Angiotensin Using Capillary Electrophoresis with Laser-Induced Fluorescence Detection

• Published in: Analytical biochemistry 1995-03, Vol.225 (2), p.341-345
• Main Author: Chen, F.T.A.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Angiotensin I - chemistry; Angiotensin I - metabolism; Angiotensin II - chemistry; Angiotensin II - metabolism; Animals; Carbocyanines - chemistry; Carboxypeptidases - metabolism; Cathepsin A; Electrophoresis - methods; Endopeptidase K; Endopeptidases - analysis; Endopeptidases - metabolism; Humans; Lasers; Molecular Sequence Data; Peptides - chemistry; Peptides - metabolism; Serine Endopeptidases - metabolism; Spectrometry, Fluorescence - methods; Trypsin
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.1995.1164

The hydrolytic cleavage of a cyanine (Cy3)-labeled angiotensin, catalyzed by various proteases, was studied by capillary electrophoresis (CE) with laser-induced fluorescence detection (LIF). The end-labeled peptides and the Cy3 diacid internal standard were separated on a 20-μm × 27-cm capillary with LIF detection (emission, 580 nm) using a frequency-doubled solid-state diode laser emitting at 532 nm or a He-Ne laser emitting at 543 nm. Hydrolysis of the Cy3-labeled angiotensin I, catalyzed by proteinase K, is a sequential process beginning from the C-terminal of the peptide, instead of from random cleavages. Trypsin catalyzes a specific cleavage of Cy3-angiotensin I to Cy3-Asp-Arg as anticipated. Using a combination of endopeptidase and carboxypeptidases, the remnant of the labeled species was characterized by CE-LIF. The method provides a general tool for studying the mechanism of protease-catalyzed hydrolysis of peptide.