Mechanism for the unfolding and refolding of ribonuclease A. Kinetic studies utilizing spectroscopic methods.
Mechanism for the unfolding and refolding of ribonuclease A. Kinetic studies utilizing spectroscopic methods.
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核糖核酸酶 A 的展开和重折叠机制。利用光谱方法进行动力学研究。
DOI：
10.1021/bi00272a007
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发表时间：
1983
期刊：
影响因子：
2.9
通讯作者：
Brandts,JF
中科院分区：
文献类型：
--
作者：
Lin,LN;Brandts,JF
材料与方法RNase购自Sigma Chemical Co. (type XII-A，批号49 C-8047）。尿素（超纯）购自施瓦茨/Mann Co.。所有其他化学品均为试剂级，并已在之前的论文中进行了描述（Lin & Brandts，1983 a，B; Lin & Brandts，1978）。RNase重折叠和解折叠的吸光度测量（手动混合）。采用配备恒温池保持器的卡里14分光光度计跟踪RNase A复性过程中吸光度变化（在287 nm处）作为时间的函数。使用灵敏的0.1-A滑线。实验在10.5和16 ℃两个温度下进行。为了进行重折叠，在存在或不存在（NH 4）2S 〇 4的情况下，将未折叠的RNA酶（在高尿素浓度下，在低pH下）稀释到乙酸钠缓冲液中至不同的最终尿素浓度，然后在观察之前用刮刀将溶液混合几秒钟。混合死区时间约为5-10 s。采用外推至零时间来获得缓慢重折叠相的总吸光度变化，发现其总是接近总吸光度变化的80%。在观察到多个动力学相的情况下，使用剥离法来分辨分离的相。（1975）用于获得RNase在高尿素溶液中展开的慢相速率。程序如下：通过移液到也在10.5 ℃的尿素溶液（170 μ，5.6M，pH 2.0）中预定的时间间隔，将10.5 ℃的H2 G（30 μ，150 mg/mL）中的少量天然RNA酶展开。然后，将167 µ溶液快速移液至含有2.5 mL 0.1 M
Materials and MethodsMaterials. RNase was purchased from Sigma Chemical Co.(type XII-A, lot 49C-8047). Urea (Ultra Pure) was obtained from Schwarz/Mann Co. All other chemical are reagent grade and have been described in previous papers (Lin & Brandts, 1983a, b; Lin & Brandts, 1978). Absorbance Measurements of RNase Refolding and Un-folding (Manual Mixing). A Cary 14 spectrophotometer equipped with a thermostated cell holder was employed to follow theabsorbance change (at 287 nm) as a function of time during RNase A refolding. The sensitive 0.1-A slide wire was used. The experiments were carried out at two temperatures, 10.5 and 16 C. For refolding, the unfolded RNase (in high urea concentration, at low pH) was diluted into sodiumacetate buffer to different final urea concentrations in thepresence or absence of (NH4) 2S04, and then the solution was mixed with a spatula for a few seconds before observation. The mixing dead time was about 5-10 s. Extrapolation to zero time was employed to obtain the total absorbance change of the slow refolding phases, which was always found to be close to 80% of total absorbance change. In cases where more than one kinetic phases were observed, the peel-off method was used to resolve the separate phases.A double-jumpmethod similar to that of Brandts et al.(1975) was used to obtain therates of the slow phases of RNase unfolding in high-urea solution. The procedures were as follows: A small amount of native RNase in H2G (30 µ, 150 mg/mL) at 10.5 C was unfolded by pipetting into a urea solution (170 µ, 5.6 M, pH 2.0) also at 10.5 C for predetermined time intervals. Then, 167 µ of solution was quickly pipetted into a quartz cuvette containing 2.5 mL of 0.1 M