Rydén L, Ofverstedt L G, Beinert H, Emptage M H, Kennedy M C
J Biol Chem. 1984 Mar 10;259(5):3141-4.
Existing estimates of the molar content of iron and labile sulfide in aconitase are varying and deviate from integral numbers. The proposed model of the iron-sulfur cluster of inactive aconitase, suggesting it to contain a single [3Fe-4S] cluster, has prompted us to reinvestigate the basic physicochemical data of the enzyme to arrive at a more precise figure of the stoichiometry of Fe and S2-. The molecular weight of aconitase estimated from low speed sedimentation equilibrium was 80,900 +/- 2,200. Gel chromatography in 6 M guanidine HCl showed the presence of a single peptide chain of 710 residues, corresponding to a Mr of 78,400, while gel electrophoresis in presence of sodium dodecyl sulfate gave a value of 83,000. Both values are in reasonable agreement with the value obtained from sedimentation equilibrium. Protein determination by amino acid analyses, together with iron and sulfur analyses of 20 different preparations of greater than or equal to 95% purity, gives values of 2.9 +/- 0.2 Fe/mol and 3.9 +/- 0.2 S2-/mol. The data obtained are thus in agreement with the [3Fe-4S] model of the iron-sulfur cluster of inactive aconitase.
目前对乌头酸酶中铁和不稳定硫化物摩尔含量的估计各不相同，且偏离整数。所提出的无活性乌头酸酶铁硫簇模型表明其含有单个[3Fe - 4S]簇，这促使我们重新研究该酶的基本物理化学数据，以得出铁和S2-化学计量的更精确数值。通过低速沉降平衡估算的乌头酸酶分子量为80,900±2,200。在6 M盐酸胍中进行凝胶色谱分析显示存在一条由710个残基组成的单肽链，对应分子量为78,400，而在十二烷基硫酸钠存在下进行凝胶电泳得到的值为83,000。这两个值与从沉降平衡获得的值合理相符。通过氨基酸分析进行蛋白质测定，以及对20种纯度大于或等于95%的不同制剂进行铁和硫分析，得到的值为2.9±0.2 Fe/mol和3.9±0.2 S2-/mol。因此，所获得的数据与无活性乌头酸酶铁硫簇的[3Fe - 4S]模型相符。
