应用免疫和质谱法对亚心形扁藻氢酶蛋白进行亚细胞定位和分类

Subcellular Localization and Identification of Hydrogenase Isolated from the Marine Green Alga Platymonas subcordiformis Using Immunoprecipitation and MALDI-TOF MS

摘要:

亚心形扁藻(Platymonas subcordiformis)是新发现的一株产氢海洋单细胞绿藻，经过胁迫调控可实现一定时间的持续产氢。氢酶是亚心形扁藻在胁迫条件下进行光合产氢的一个关键酶。但到目前为止，亚心形扁藻氢酶相关信息仍不清楚。利用蛋白合成抑制剂氯霉素和放线菌酮对亚心形扁藻氢酶活性进行考察，同时利用免疫印迹技术和免疫胶体金电镜对亚心形扁藻氢酶蛋白进行亚细胞定位分析。结果表明：亚心形扁藻氢酶蛋白可能由胞浆内合成，在叶绿体行使功能。采用免疫共沉淀技术富集亚心形扁藻细胞氢酶蛋白，SDS-聚丙烯酰胺凝胶电泳（SDS－PAGE）对免疫共沉淀复合物进行分离，从胶中切取目的蛋白条带，胶内酶解后进行基质辅助激光解吸飞行时间质谱（MALDI-TOF-MS）分析，得到相应的肽指纹图谱，通过搜索数据库检索初步断定亚心形扁藻氢酶蛋白为铁氢酶。

Abstract:

A marine unicellular green alga, Platymonas subcordiformis, was demonstrated to photobiologically produce hydrogen gas from seawater. The objective of this study was to localize and identify the hydrogenase isolated from P. subcordiformis. Adaptation in the presence of inhibitors of protein biosynthesis indicated that the hydrogenase was much more inhibited by cycloheximide than that by chloramphenicol. The result suggested that the hydrogenase isolated from P. subcordiformis is probably synthesized in cytoplasmic ribosomes. Both Western blot analysis and immunogold electron microscopy demonstrate that theP. subcordiformis hydrogenase is mainly located in the chloroplast stroma. The proteins that reacted specifically with the antibodies against the iron hydrogenase isolated from Chlamydomonas reinhardtii were concentrated by immunoprecipitation. The separated protein bands were cut out of the SDS-PAGE gel, in-gel digested by trypsin, and analyzed by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). Mascot was employed for analysis of the MALDI data using the public databases NCBInr. The hydrogenase isolated from P. subcordiformis was identified to be the Fe-hydrogenase.