Irvine G B, Ennis M, Williams C H
Division of Biochemistry, School of Biology and Biochemistry, Queen's University of Belfast, Northern Ireland, United Kingdom.
Anal Biochem. 1990 Mar;185(2):304-7. doi: 10.1016/0003-2697(90)90298-n.
A simple, inexpensive, and sensitive assay for peptidase activity has been devised. The assay was performed in a microtiter plate and was based on fluorogenic peptide substrates, many of which are commercially available. 7-Amino-4-methyl coumarin the fluorescent product liberated during an incubation period of between 1 and 16 h, was detected by inspection of the plate under ultraviolet light of wavelength 356 nm. A fluorometer was not required. Using alpha-chymotrypsin as a model enzyme, with succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine 4-methyl-coumaryl-7-amide as substrate, it was shown that as little as 4 fmol of enzyme could be detected. The method was non-quantitative and was particularly suited to location of enzyme activity in fractions during a purification procedure. The validity of the assay was demonstrated by detection of activity of a known enzyme, alpha-chymotrypsin, after its purification by size-exclusion high-performance liquid chromatography. The method was used to locate two forms of aminopeptidase activity, in fractions from size-exclusion chromatography of an extract from reproductive tissue of Helix aspersa, using L-leucine 4-methyl-coumaryl-7-amide as substrate.
已设计出一种用于肽酶活性的简单、廉价且灵敏的检测方法。该检测在微量滴定板中进行，基于荧光肽底物，其中许多在市面上都可买到。7-氨基-4-甲基香豆素是在1至16小时的孵育期内释放出的荧光产物，通过在波长356nm的紫外光下检查平板来检测。不需要荧光计。以α-胰凝乳蛋白酶作为模型酶，使用琥珀酰-L-丙氨酰-L-丙氨酰-L-脯氨酰-L-苯丙氨酸4-甲基香豆素-7-酰胺作为底物，结果表明可检测到低至4飞摩尔的酶。该方法是非定量的，特别适用于在纯化过程中定位各组分中的酶活性。通过在经尺寸排阻高效液相色谱纯化后的已知酶α-胰凝乳蛋白酶中检测活性，证明了该检测方法的有效性。该方法用于定位两种氨肽酶活性形式，使用L-亮氨酸4-甲基香豆素-7-酰胺作为底物，从光滑双脐螺生殖组织提取物的尺寸排阻色谱分离组分中进行检测。
