Radyukhin Victor A, Serebryakova Marina V, Ksenofontov Alexander L, Lukashina Elena V, Baratova Lyudmila A
A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Leninskie Gory, Moscow 119992, Russia.
Protein Pept Lett. 2006;13(9):907-13. doi: 10.2174/092986606778256207.
A method of isolation of hydrophobic membrane-bound C-terminal domain of influenza virus A hemagglutinin (HA) is suggested. The method is based on the insertion of HA into octylglucoside micelles followed by pepsin or thermolysin hydrolysis. Subsequent treatment of proteolytic digests with chloroform-hexafluoroisopropanol mixture resulted in the extraction of a few hydrophobic peptides into organic phase. Mass-spectrometry (MALDI-TOF) analysis revealed that the peptides with ion masses corresponding to the anchoring C-terminal domain with or without modifications predominated in the organic solution. The data obtained confirmed our speculation on the possibility of the suggested isolation scheme following from the strong interactions of anchoring domains in compact trimeric structure of HA spikes combined with micelle protection effect. Several appropriate peptides presence in the organic phase apparently arises from the presence of a few accessible proteolytic sites in HA transmembrane region.
本文提出了一种分离甲型流感病毒血凝素（HA）疏水膜结合C末端结构域的方法。该方法基于将HA插入辛基葡糖苷胶束中，然后用胃蛋白酶或嗜热菌蛋白酶进行水解。随后用氯仿 - 六氟异丙醇混合物处理蛋白水解消化物，导致一些疏水肽被萃取到有机相中。基质辅助激光解吸电离飞行时间质谱（MALDI - TOF）分析表明，在有机溶液中，离子质量对应于有或没有修饰的锚定C末端结构域的肽占主导地位。获得的数据证实了我们的推测，即由于HA刺突紧密三聚体结构中锚定结构域的强相互作用以及胶束保护作用，所建议的分离方案具有可行性。有机相中存在几种合适的肽显然是由于HA跨膜区域存在一些可及的蛋白水解位点。
