一种以三肽L-脯氨酰-L-脯氨酰-L-丙氨酸为底物的二肽基肽酶IV荧光新检测法。

A new fluorescence assay for dipeptidylpeptidase IV using tripeptide L-prolyl-L-prolyl-L-alanine as substrate.

作者信息

Iijima K I, Takei T, Hino M, Hayakawa T

出版信息

J Biochem Biophys Methods. 1980 Aug;3(2):89-96. doi: 10.1016/0165-022x(80)90031-7.

Abstract

We have developed a new fluorescence assay for dipeptidylpeptidase IV using a tripeptide, L-prolyl-L-prolyl-L-alanine, which might be one of the potential natural substrates. The principle of the assay is based on the measurement of fluorescent adduct between alanine liberated from the tripeptide by enzymatic hydrolysis and o-phthaldialdehyde in the presence of 2-mercaptoethanol in aqueous alkaline medium. This new assay is sensitive enough to measure the enzyme activity in as little as 0.01 microliter of human serum and in crevicular fluid obtained from human gingival sulcus. The Km value for the tripeptide was 1.7 x 10(-5) M which is less than one-tenth of that obtained with other chromogenic or fluorogenic substrates. The interference by serum was overcome by simply incorporating the same amount of serum in the standards.

摘要

我们使用三肽L-脯氨酰-L-脯氨酰-L-丙氨酸开发了一种用于二肽基肽酶IV的新型荧光测定法，该三肽可能是潜在的天然底物之一。该测定法的原理基于在水性碱性介质中，在2-巯基乙醇存在下，通过酶促水解从三肽中释放的丙氨酸与邻苯二甲醛之间荧光加合物的测量。这种新的测定法灵敏度足够高，能够测量低至0.01微升人血清和从人牙龈沟获得的龈沟液中的酶活性。该三肽的Km值为1.7×10⁻⁵ M，小于使用其他显色或荧光底物获得的值的十分之一。通过在标准品中简单加入相同量的血清，克服了血清的干扰。