Smith G K, D'Alessio G, Schaffer S W
Biochemistry. 1978 Jun 27;17(13):2633-8. doi: 10.1021/bi00606a027.
Totally reduced and denatured seminal ribonuclease was regenerated using the glutathione redox system. The refolding kinetics of the enzyme were determined as a function of redox state, temperature from 14 to 43 degrees C, pH, and protein concentration. The maximal rate of regeneration occurred with 3 x 10(-3) M reduced glutathione, 6 x 10(-4) M oxidized glutathione, 24 to 30 degrees C, and pH 8.2. The products of the refolding process were characterized by Sephadex G-75, sodium dodecyl sulfate gel electrophoresis, enzymatic activity, circular dichroism, and amino acid analysis. The results indicate that the native dimeric form of the enzyme is not produced during refolding to any appreciable extent; rather, the major product is monomeric. The purified monomer exhibits twice the activity of the native enzyme toward yeast RNA. Its circular dichroism spectrum is different from the native enzyme and is quite similar to that of pancreatic ribonuclease A. Amino acid analyses showed that two glutathione molecules are bound to the monomer, suggesting that cysteine-31 and -32, which normally form the intermolecular disulfide bonds, are blocked.
利用谷胱甘肽氧化还原系统使完全还原和变性的精浆核糖核酸酶复性。测定了该酶的复性动力学与氧化还原状态、14至43摄氏度的温度、pH值以及蛋白质浓度的函数关系。在3×10⁻³M还原型谷胱甘肽、6×10⁻⁴M氧化型谷胱甘肽、24至30摄氏度以及pH 8.2的条件下，复性速率达到最大值。通过葡聚糖凝胶G - 75、十二烷基硫酸钠凝胶电泳、酶活性、圆二色性以及氨基酸分析对复性过程的产物进行了表征。结果表明，在复性过程中并未大量产生该酶的天然二聚体形式；相反，主要产物是单体。纯化后的单体对酵母RNA的活性是天然酶的两倍。其圆二色性光谱与天然酶不同，与胰腺核糖核酸酶A的光谱非常相似。氨基酸分析表明，两个谷胱甘肽分子与单体结合，这表明通常形成分子间二硫键的半胱氨酸 - 31和 - 32被阻断。
