棕色固氮菌突变种UW3部分提纯的固氮酶CrFe蛋白溶液中残存细菌铁蛋白的晶体生长及鉴定

从无钼、无氨而含铬的固氮培养基中生长的棕色固氮菌(Azotobacter vinelandii Lipmann)突变种UW3中纯化得到了部分纯的CrFe蛋白.在试图培养CrFe蛋白大晶体时发现,棕色晶体和砖红色晶体可同时或单独出现.SDS-PAGE和厌氧天然PAGE皆表明,棕色晶体主要由与固氮酶钼铁蛋白(Av1)类似大小的亚基(～60 kD)组成,而砖红色晶体则由～20kD亚基组成.免疫分析表明只有～60kD的亚基可与固氮酶钼铁蛋白的抗体反应,而～20kD亚基则无这种反应.在部分纯的CrFe蛋白溶液中,～20 kD的总蛋白含量远低于～60 kD蛋白的含量,表明由这种小亚基组成的蛋白只是CrFe蛋白溶液中的一种污染蛋白.用3,5-二氨基苯甲酸染色的天然电泳表明,形成砖红色和棕色晶体的蛋白是迁移率不同的两种含铁蛋白.质谱分析表明砖红色晶体蛋白为棕色固氮菌的细菌铁蛋白.分辨率为2.34 A的X射线衍射结果也表明,砖红色晶体属于H3空间群,晶胞参数为a=124.965A,b=124.965A和c=287.406 A.即将发表的三维结构解析表明,此砖红色晶体确为24聚体的细菌铁蛋白.

Crystal Growth and Characterization of Residual Bacterioferritin in Partially Purified Nitrogenase CrFe Protein Solution from a Mutant UW3 of Azotobacter vinelandii

While attempting to obtain large crystals of nitrogenase CrFe protein, brown crystals and brick red crystals were simultaneously or independently obtained from CrFe protein preparation, which was partially purified from a mutant UW3 of Azotobacter vinelandii Lipmann grown on Mo-, ammonia-free but Cr-containing medium. SDS-PAGE and anoxic native-PAGE analysis consistently showed that the protein of the brown crystal was mainly composed of subunits (～60 kD) similar to those of Av1 (MoFe protein), while the protein of the brick red crystal was composed of ～20 kD subunits. And only the larger subunits rather than the smaller ones were detectable by Western blot to the antibody of Av1. Comparing with the large subunits, the amount of the small subunits in the partially purified CrFe protein solution was much smaller, indicating that the protein composed of the smaller subunits was one of contamination proteins for CrFe protein. Detection by 3, 5-diaminobenzoic acid of native-PAGE gels showed that the proteins forming the brick red crystal and the brown crystal were two kinds of iron-containing proteins with different electrophoretic mobility on the gel. The analysis of matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) proved that the protein forming the brick red crystal was bacterioferritin of A. vinelandii (AvBF). X-ray diffraction to 2.34 Å resolution showed that the crystal belonged to space group H3, with unit-cell parameters a = 124.965 Å, b=124.965 Å and c = 287.406 Å. The detailed structural analysis published in the near future has confirmed that the brick red crystal is that of 24-meric bacterioferritin.