Fink A L, Anderson W D, Hattersley J E, Lustig B S
Department of Chemistry, University of California, Santa Cruz, 95064.
FEBS Lett. 1988 Aug 15;236(1):190-4. doi: 10.1016/0014-5793(88)80312-0.
Unfolded ribonuclease A consists of 20% fast refolding (Uf) and 80% slow refolding material (Us). The latter consists of at least two different forms which refold at different rates. We have used absorbance and fluorescence spectrophotometry to compare the kinetics of refolding in aqueous and aqueous-methanol solutions. At 1 degree C and pH 3.0, the addition of increasing concentrations of methanol (to 50%, v/v) had negligible effect on the rates and amplitudes of the slow refolding Us states. The effect of temperature on the two slow phases of refolding was determined in 35 and 50% methanol. From Arrhenius plots the energies of activation were found to be in the vicinity of 20 kcal/mol for both processes. The results suggest that both slow phases correspond to proline isomerization, and that the presence of methanol does not significantly perturb the overall refolding process. It is possible that the faster of the slow refolding phases corresponds to the isomerization of a proline residue which is trans in the folded native state but which undergoes extensive isomerization to the cis conformation in the unfolded state.
未折叠的核糖核酸酶A由20%的快速重折叠物质（Uf）和80%的慢速重折叠物质（Us）组成。后者至少由两种以不同速率重折叠的不同形式组成。我们使用吸光度和荧光分光光度法来比较在水溶液和水 - 甲醇溶液中的重折叠动力学。在1℃和pH 3.0条件下，加入浓度不断增加的甲醇（至50%，v/v）对慢速重折叠Us状态的速率和幅度影响可忽略不计。在35%和50%甲醇中测定了温度对重折叠的两个慢相的影响。从阿累尼乌斯图中发现，两个过程的活化能都在20千卡/摩尔左右。结果表明，两个慢相都对应于脯氨酸异构化，并且甲醇的存在不会显著干扰整体重折叠过程。有可能较慢的慢速重折叠相对应于脯氨酸残基的异构化，该脯氨酸残基在折叠的天然状态下是反式的，但在未折叠状态下会大量异构化为顺式构象。
