Venkatesh Y P, Vithayathil P J
Int J Pept Protein Res. 1984 May;23(5):494-505. doi: 10.1111/j.1399-3011.1984.tb02750.x.
The isolation and characterization of the initial intermediates formed during the irreversible acid denaturation of enzyme Ribonuclease A are described. The products obtained when RNase A is maintained in 0.5 M HCl at 30 degrees for periods up to 20 h have been analyzed by ion-exchange chromatography on Amberlite XE-64. Four distinct components were found to elute earlier to RNase A; these have been designated RNase Aa2, Aa1c, Aa1b, and Aa1a in order of their elution. With the exception of RNase Aa2, the other components are nearly as active as RNase A. Polyacrylamide gel electrophoresis at near-neutral pH indicated that RNase Aa1a, Aa1b, and Aa1c are monodeamidated derivatives of RNase A; RNase Aa1c contains, in addition, a small amount of a dideamidated component. RNase Aa2, which has 75% enzymic activity as compared to RNase A, consists of dideamidated and higher deamidated derivatives of RNase A. Except for differences in the proteolytic susceptibilities at an elevated temperature or acidic pH, the monodeamidated derivatives were found to have very nearly the same enzymic activity and the compact folded structure as the native enzyme. Fingerprint analyses of the tryptic peptides of monodeamidated derivatives have shown that the deamidations are restricted to an amide cluster in the region 67-74 of the polypeptide chain. The initial acid-catalyzed deamidation occurs in and around the 65-72 disulfide loop giving rise to at least three distinct monodeamidated derivatives of RNase A without an appreciable change in the catalytic activity and conformation of the ribonuclease molecule. Significance of this specific deamidation occurring in highly acidic conditions, and the biological implications of the physiological deamidation reactions of proteins are discussed.
本文描述了在核糖核酸酶A不可逆酸变性过程中形成的初始中间体的分离和特性鉴定。当核糖核酸酶A在0.5M盐酸中于30℃保持长达20小时时所获得的产物，已通过在Amberlite XE - 64上进行离子交换色谱分析。发现有四种不同的组分比核糖核酸酶A更早洗脱；按照洗脱顺序，它们被命名为核糖核酸酶Aa2、Aa1c、Aa1b和Aa1a。除核糖核酸酶Aa2外，其他组分的活性几乎与核糖核酸酶A相同。近中性pH条件下的聚丙烯酰胺凝胶电泳表明，核糖核酸酶Aa1a、Aa1b和Aa1c是核糖核酸酶A的单脱酰胺衍生物；核糖核酸酶Aa1c还含有少量的双脱酰胺组分。与核糖核酸酶A相比具有75%酶活性的核糖核酸酶Aa2，由核糖核酸酶A的双脱酰胺和更高程度脱酰胺的衍生物组成。除了在高温或酸性pH下蛋白水解敏感性的差异外，发现单脱酰胺衍生物具有与天然酶非常接近的酶活性和紧密折叠结构。单脱酰胺衍生物胰蛋白酶肽段的指纹分析表明，脱酰胺作用局限于多肽链67 - 74区域的酰胺簇。最初的酸催化脱酰胺作用发生在65 - 72二硫键环及其周围，产生至少三种不同的核糖核酸酶A单脱酰胺衍生物，而核糖核酸酶分子的催化活性和构象没有明显变化。本文讨论了在高酸性条件下发生的这种特定脱酰胺作用的意义以及蛋白质生理脱酰胺反应的生物学意义。
