Li W, Churchich J E
Department of Biochemistry & Cellular & Molecular Biology, University of Tennessee, Knoxville, USA.
Eur J Biochem. 1997 May 15;246(1):127-32. doi: 10.1111/j.1432-1033.1997.t01-1-00127.x.
CD spectroscopy reveals that mitochondrial malate dehydrogenase in 3M guanidinium chloride shows little residual secondary structure. Refolding of the denatured protein by dilution with buffer of pH 7.5 does not restore the CD spectrum of the native enzyme. A partially folded intermediate, possessing 25% of the alpha-helix content of the native enzyme, is formed upon dilution. The partially folded intermediate binds the extrinsic probe 1-anilinonaphtalene-8-sulfonate, and the increase in fluorescence (tenfold) is accompanied by a blue shift in the band position of the emission spectrum. Partially folded malate dehydrogenase is devoid of catalytic activity. In vitro refolding of the denatured protein takes place in the presence of dithiotreitol and thioredoxin. In the presence of micromolar concentrations of thioredoxin, a recovery of approximately 70% of the catalytic activity was observed. Emission-anisotropy titrations of oxidized thioredoxin, tagged with a fluorescent probe, revealed that the oxidoreductase recognizes partially folded intermediates of malate dehydrogenase with a dissociation constant of 6 microM. Moreover, a covalently linked complex formed by thioredoxin and monomeric malate dehydrogenase was detected by SDS/PAGE. A general mechanism is postulated for the reactivation of denatured proteins by thioredoxin.
圆二色光谱显示，在3M氯化胍中的线粒体苹果酸脱氢酶几乎没有残留的二级结构。用pH 7.5的缓冲液稀释使变性蛋白复性，不能恢复天然酶的圆二色光谱。稀释时会形成一种部分折叠的中间体，其α-螺旋含量为天然酶的25%。该部分折叠的中间体结合外在探针1-苯胺基萘-8-磺酸盐，荧光增强（十倍），同时发射光谱的谱带位置发生蓝移。部分折叠的苹果酸脱氢酶没有催化活性。变性蛋白的体外复性在二硫苏糖醇和硫氧还蛋白存在的情况下进行。在微摩尔浓度的硫氧还蛋白存在下，观察到催化活性恢复了约70%。用荧光探针标记的氧化型硫氧还蛋白的发射各向异性滴定显示，氧化还原酶识别苹果酸脱氢酶的部分折叠中间体，解离常数为6 microM。此外，通过SDS/PAGE检测到硫氧还蛋白与单体苹果酸脱氢酶形成的共价连接复合物。推测了硫氧还蛋白使变性蛋白重新活化的一般机制。
