用于实时监测蛋白质水解的新型简易荧光蛋白酶测定法

IF 4.6 Q2 MATERIALS SCIENCE, BIOMATERIALS
Miha Bahun , Nataša Poklar Ulrih
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引用次数: 0

摘要

测量蛋白酶的活性对于研究这些酶的生理功能和商业应用至关重要。与众多基于发色性或荧光性肽底物的蛋白酶检测方法相比,目前还缺乏实时监测蛋白质降解的方法。在这里,我们报告了一种蛋白酶检测方法,它采用 SYPRO Orange 作为荧光探针来跟踪蛋白水解过程。我们使用四种不同的蛋白质底物,对两种热稳定性不同的亚铁酶进行了测定,证明了该测定法的功能。该检测方法与实时 PCR 仪器兼容,即使在高温条件下也能对低容量样品进行连续荧光测量。因此,该检测方法特别适用于对热稳定性蛋白酶进行高通量表征。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

A novel simple fluorometric protease assay for monitoring hydrolysis of proteins in real time

A novel simple fluorometric protease assay for monitoring hydrolysis of proteins in real time
Measuring the activity of proteases is essential for investigating both the physiological functions and commercial applications of these enzymes. In contrast to the numerous protease assays that are based on chromogenic or fluorogenic peptide substrates, there is a lack of approaches to monitor degradation of proteins in real time. Here we report a protease assay where SYPRO Orange is employed as a fluorogenic probe to follow proteolysis. The functionality of the assay was demonstrated with the two subtilases of varying thermostability, using four different protein substrates. The assay is compatible with a real-time PCR instrument which allows continuous fluorescence measurements in low-volume samples even at high temperatures. This makes the assay especially suitable for high-throughput characterization of thermostable proteases.
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来源期刊
ACS Applied Bio Materials
ACS Applied Bio Materials Chemistry-Chemistry (all)
CiteScore
9.40
自引率
2.10%
发文量
464
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