Oertle M, Filipovic D, Richter C, Winterhalter K H, Di Iorio E E
Laboratorium fuer Biochemie I, ETH Zentrum, Zurich, Switzerland.
Arch Biochem Biophys. 1991 Nov 15;291(1):24-30. doi: 10.1016/0003-9861(91)90100-w.
A procedure for the isolation of native proteins from membranes by isoelectric focusing is described. It was used to resolve into six components the major fraction of cytochrome P450, obtained from liver microsomes of phenobarbital-treated rats, after chromatography on DE-52 cellulose. When eluted from the gel, these proteins are in a native form as shown by (a) the light absorption spectra of the Soret region of their reduced carbonyl derivatives, all characterized by maxima around 450 nm, and (b) their enzymatic activities toward three different substrates. Characterization by a monoclonal antibody and partial sequence analysis of tryptic peptides reveal that three of the IEF-purified proteins have P450IIB1 character, whereas the other three are related to P450IIB2.
本文描述了一种通过等电聚焦从膜中分离天然蛋白质的方法。该方法用于将经苯巴比妥处理的大鼠肝脏微粒体在DE-52纤维素上色谱分离后获得的细胞色素P450主要部分解析为六个组分。从凝胶上洗脱后，这些蛋白质呈天然形式，这表现为：(a) 其还原羰基衍生物的Soret区域的光吸收光谱，所有光谱的特征峰均在450 nm左右；(b) 它们对三种不同底物的酶活性。通过单克隆抗体进行的表征以及胰蛋白酶肽段的部分序列分析表明，等电聚焦纯化的蛋白质中有三种具有P450IIB1特征，而另外三种与P450IIB2相关。
