Fedorova N V, Ksenofontov A L, Viryasov M B, Baratova L A, Timofeeva T A, Zhirnov O P
A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia.
J Chromatogr B Biomed Sci Appl. 1998 Feb 27;706(1):83-9. doi: 10.1016/s0378-4347(97)00558-6.
The M1 protein of influenza virus is a highly hydrophobic polypeptide that is resistant to enzyme cleavage during incubation in water solutions. We show here that the M1 protein that is immobilized on an insoluble activated support (thiopropyl Sepharose-6B) by means of a thiol-disulfide exchange reaction acquires sensitivity to trypsin. After tryptic digestion noncysteine-containing peptides of M1 were removed by washing the support, while cysteine-containing ones were detached from the support by reduction. As a result, 24 unique tryptic peptides of M1 protein were clearly separated by reversed-phase high-performance liquid chromatography. The described method opens a new way to the investigation of functional properties of distinct domains of viral thiol proteins.
流感病毒的M1蛋白是一种高度疏水的多肽，在水溶液中孵育时对酶切具有抗性。我们在此表明，通过硫醇-二硫键交换反应固定在不溶性活化支持物（硫丙基琼脂糖-6B）上的M1蛋白对胰蛋白酶变得敏感。胰蛋白酶消化后，通过洗涤支持物去除M1的不含半胱氨酸的肽段，而含半胱氨酸的肽段则通过还原从支持物上分离下来。结果，通过反相高效液相色谱法清晰地分离出了M1蛋白的24个独特的胰蛋白酶肽段。所描述的方法为研究病毒硫醇蛋白不同结构域的功能特性开辟了一条新途径。
