AUT凝胶电泳在牛胰核糖核酸酶折叠研究中的应用

Application of Acetic Acid-Urea-Triton Polyacrylamide Gel Electrophoresis in the Study of Bovine Pancreatic Ribonuclease A Folding

目的:牛胰核糖核酸酶是一种用于蛋白折叠研究的经典模式蛋白,在折叠研究过程中主要使用高效液相色谱用于分离检测不同阶段的蛋白折叠中间体。高效液相色谱具有自动化、分离效果好、样品可回收等优点,同时也存在检测通量较低、仪器设备较为昂贵等不足。AUT凝胶电泳简便、快捷、检测通量较高,本文尝试将其应用于牛胰核糖核酸酶的折叠研究。方法:使用AUT凝胶电泳、酶活性检测、质谱对牛胰核糖核酸酶还原变性过程及产生的折叠中间体进行检测;通过高效液相色谱和质谱对折叠中间单体进行分离检测,并分别进行AUT凝胶电泳检测以解析各折叠中间单体在电泳中的条带位置;通过AUT凝胶电泳和酶切后质谱鉴定各折叠中间单体的二硫键配对方式。结果:AUT凝胶电泳可以有效区分不同条件下的牛胰核糖核酸酶还原变性过程,检测结果与酶活性、质谱结果相符,并可以很好地区分牛胰核糖核酸酶还原变性过程折叠中间体。高效液相色谱将牛胰核糖核酸酶还原变性过程折叠中间体分离为13个色谱峰,并与AUT凝胶电泳中的11个条带位置进行匹配。确认牛胰核糖核酸酶还原变性过程折叠中间单体的二硫键配对方式,并与AUT凝胶电泳条带进行匹配,Cys58-Cys110和Cys26-Cys84构象熵减作用强于Cys40-Cys95和Cys65-Cys72。结论:AUT凝胶电泳适用于检测牛胰核糖核酸酶折叠中间体,可以与高效液相色谱、质谱等检测技术相互补充,共同应用于牛胰核糖核酸酶的折叠研究。

Objective： Bovine pancreatic ribonuclease A（RNase A） is widely applied as a benchmark protein in folding studies for its stable structure and easy renaturation. And high performance liquid chromatography（HPLC） is commonly used in the research on RNase A folding for its advantage of automation, effective separation and sample recycling. However, with the deficiency of low-throughput detection and high-cost equipment, the application of HPLC is limited to a certain extent in the study of RNase A folding.In this study we will introduce acetic acid-urea-Triton polyacrylamide（AUT） gel electrophoresis as a new simple technology into RNase A folding. Methods： Reductive unfolding of RNase A and the folding intermediates with native disulfide bonds were detected by AUT gel electrophoresis, enzyme activity and mass spectrometry. Intermediate monomers were separated and identified by HPLC and mass spectrometry, respectively, followed by detection of AUT gel electrophoresis to match band positions. And the native disulfide pairing of intermediate monomers were determined by detection of AUT gel electrophoresis to the partial reduction of 3 ss with native disulfide bonds and of mass spectrometry to trypsin digestion products of intermediate monomers. Results： AUT gel electrophoresis could monitor the whole process of reductive unfolding of RNase A, consistent with the result of enzyme activity and mass spectrometry, and well distinguish disulfide-bonded intermediates of RNase A folding based on their difference in compactness caused by disulfide bonds and effective charges. Intermediates with native disulfide bonds can be divided into 13 chromatographic peaks by HPLC, and matched to 11 bands detected by AUT gel electrophoresis. Furthermore, disulfide pairing of intermediate monomers were identified completely and matched to bands of AUT gel electrophoresis, which referred that entropy decrease caused by Cys58-Cys110 or Cys26-Cys84 is larger than that by Cys40-Cys95 or Cys65-Cys72. Conclusions： As a relatively simple, rapid, accurate and economical technology, AUT gel electrophoresis is competent for the separation of folding intermediates, complemented with HPLC and mass spectrometry, and must bepromising in the study of RNase A folding.