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, 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-microns x 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.
Chen
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Advanced Technology Center, Beckman Instruments Inc., Fullerton, California 92634, USA.
D000595:Amino Acid Sequence; D000803:Angiotensin I / Q000737:chemistry / Q000378:metabolism; D000804:Angiotensin II / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002232:Carbocyanines / Q000737:chemistry*; D002268:Carboxypeptidases / Q000378:metabolism; D043402:Cathepsin A; D004586:Electrophoresis / Q000379:methods*; D019286:Endopeptidase K; D010450:Endopeptidases / Q000032:analysis* / Q000378:metabolism*; D006801:Humans; D007834:Lasers; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D014357:Trypsin
D002232:Carbocyanines; D010455:Peptides; D000804:Angiotensin II; D000803:Angiotensin I; C025427:carboxypeptidase P; D002268:Carboxypeptidases; D010450:Endopeptidases; C468629:CTSA protein, human; D043402:Cathepsin A; D012697:Serine Endopeptidases; D014357:Trypsin; D019286:Endopeptidase K