Chen F T
Advanced Technology Center, Beckman Instruments Inc., Fullerton, California 92634, USA.
Anal Biochem. 1995 Mar 1;225(2):341-5. 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-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.
采用毛细管电泳（CE）结合激光诱导荧光检测（LIF）技术，研究了各种蛋白酶催化的花菁（Cy3）标记的血管紧张素的水解裂解反应。使用发射波长为532nm的倍频固态二极管激光器或发射波长为543nm的氦氖激光器，在20微米×27厘米的毛细管上对末端标记的肽段和Cy3二酸内标进行LIF检测（发射波长580nm）分离。蛋白酶K催化的Cy3标记的血管紧张素I的水解是一个从肽段C末端开始的连续过程，而非随机裂解。正如预期的那样，胰蛋白酶催化Cy3-血管紧张素I特异性裂解为Cy3-天冬氨酰-精氨酸。通过使用内肽酶和羧肽酶的组合，利用CE-LIF对标记物的残余物进行了表征。该方法为研究蛋白酶催化肽水解的机制提供了一种通用工具。
