Assay of trypsin activity by capillary isoelectric focusing with laser-induced fluorescence detection
Assay of trypsin activity by capillary isoelectric focusing with laser-induced fluorescence detection
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DOI：
10.1002/elps.1150191308
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发表时间：
1998-10-01
期刊：
影响因子：
2.9
通讯作者：
Kasai, K
中科院分区：
文献类型：
--
作者：
Shimura, K;Matsumoto, H;Kasai, K
毛细管等电聚焦是一种高效的蛋白质分离方法，因为在分离过程中聚焦是蛋白质pI值的函数。这种技术对某些类型的肽也很有效。荧光标记和随后的激光诱导荧光检测进一步提高了这种技术的灵敏度。本文证明了这种技术在酶测定中的实用性。用丽丝胺罗丹明B的碘乙酰基衍生物在半胱氨酸残基的巯基上标记合成的九肽H-Gly-Cys-His-Glu-Ala-Arg-Ala-Glu-Glu-OH作为胰蛋白酶的底物。胰蛋白酶催化标记底物的Arg-Ala带的裂解，其集中在pH 4.8，并释放缩短的标记产物，H-Gly-*Cys-His-Glu-Ala-Arg-OH，其集中在pH 6.9（* 表示标记）。用氦氖激光器的绿色线激发，在590 nm处进行荧光检测，测定3-300 pM的产物肽，相对标准偏差为5.5%（n = 5）。在37 ℃下将胰蛋白酶与底物孵育10 min，可测定50-250 pg胰蛋白酶，相对标准偏差为5.3%（n = 5）。
Capillary isoelectric focusing is a highly effective method for the separation of proteins due to focusing as a function of their pI values in the separation process. This technique is also effective for certain types of peptides that focus well. Fluorescence labeling and subsequent detection by laser-induced fluorescence farther enhance the sensitivity of this technique. This paper demonstrates the utility of this technique in an enzyme assay. A synthetic nona peptide, H-Gly-Cys-His-Glu-Ala-Arg-Ala-Glu-Glu-OH, was labeled with an iodoacetyl derivative of Lissamine rhodamine B at the thiol group of the cysteine residue as a substrate for trypsin. Trypsin catalyzed the cleavage of the Arg-Ala band of the labeled substrate, which focused at pH 4.8, and liberated a shortened, labeled product, H-Gly-*Cys-His-Glu-Ala-Arg-OH that focused at pH 6.9 (* indicates the label). The product peptide at 3-300 pM was determined with a relative standard deviation of 5.5% (n = 5) by fluorescence detection at 590 nm with excitation by a green line of He-Ne laser. Incubation of trypsin with the substrate for 10 min at 37 degrees C allowed the determination of 50-250 pg of trypsin, with a relative standard deviation of 5.3% (n = 5).