【摘要】 棕色固氮菌固氮酶钼铁蛋白结晶,经处理先后出现一个与福林试剂呈颜色反应的洗脱峰Ⅰ和一个与茚三酮试剂呈颜色反应的洗脱峰Ⅱ。洗脱峰Ⅰ、Ⅱ均含有钼和铁。钼和铁的洗脱峰位基本上与蛋白或肽的洗脱峰位相符合。 两个洗脱峰中的成分都能激活棕色固氮菌突变株UW45无细胞抽出物的非活性组分Ⅰ,比活分别为17.8和6.8毫微克分子乙烯/分/毫微克原子钼,并能在硼氢化钾存在下自身催化还原乙炔,比活分别为16.9和50.9毫微克分子乙烯/分/毫微克原子钼。 峰Ⅰ与峰Ⅱ的组成不同,峰Ⅰ是一种组分,峰Ⅱ是两种组分的混合物。峰Ⅰ组分是不同于固氮酶钼铢蛋白的一种蛋白质。洗脱峰Ⅰ组分的分子量是5000道尔顿,洗脱峰Ⅱ两种组分的分子量分别是2100和1850道尔顿。经测定,峰Ⅰ组分和峰Ⅱ中分子量为2100道尔顿的组分含有钼、铁。因此,通过酒石酸处理可以从上清液中得到两种含钼铁的组分。这两种组分的红外吸收光谱也是明显不同的。 酒石酸处理后的沉淀再用N-甲基甲酰胺抽提获得的组分,具有恢复棕色固氮菌突变株UW45乙炔还原活性的能力。经纸层析鉴定,与Shah法制备的铁钼辅因子相类似。更多还原

【Abstract】 The crystalline MoFe-protein of nitrogenase from Azotobacter vinelandii was treated with 0.5 M tartaric acid. After neutralization followed by centrifugation, the supernatant was subjected to Sephadex G 25 column chromatography. The peak Ⅰ giving coloration with Folin reagent and the peak Ⅱ giving coloration with ninhydrin reagent appeared successively. They contained molybdenum and iron. The elution peaks of molybdenum and iron corresponded closely with those of protein or peptide.The components of the two elution peaks were able to activate the inactive component Ⅰ in extracts of mutant strain UW 45, giving a specific activity of 17.8 and 6.8 n moles C₂H₄ per min. per n mole Mo respectively. They could also. self-catalyze the acetylene reduction in the presence of KBH₄, giving a specific activity of 16.9 and 50.9 nmoles C₂H₄ per min. per n mole Mo respectively.Identification by paper chromatography, thin layer chromatography and isoelectric focusing in polyacrylamide gel demonstrated that the components of peak Ⅰ and peak Ⅱ were distinguishable. Peak Ⅰ consisted of one component while peak Ⅱ was a mixture of two. The component of peak Ⅰ was identified as a protein which differed from nitrogenase MoFe-protein by further determination with polyacrylamide gel electrophoresis and high pressure paper chromatography.By Sephadex column chromatography estimations, the molecular weight of the component of elution peak Ⅰ was 5000 daltons and those of the components of elution peak Ⅱ were 2100 and 1850 daltons respectively. The component of elution peak Ⅰ and the 2100 daltons component of elution peak Ⅱ contained Mo and Fe. Thus it might be suggested that two Mo- and Fe-containing components were obtained from the snpernatant by treatment with tartaric acid. The infra-red absorption spectra of these two components were apparently different.The preciptate formed after treatment with tartaric acid was further extracted with N-methyl formamide, the component thus obtained which was active in restoring the acetylene reduction activity of extract of A. vinelandii mutant strain UW 45 was shown to be similar to the FeMoco prepared according to the method described by Shah by paper chromatographic determination.更多还原