NMR-STUDY OF HISTIDINE RESIDUES OF S-PEPTIDE AND S-PROTEIN AND KINETICS OF H-1-H-2 EXCHANGE OF RIBONUCLEASE-A
NMR-STUDY OF HISTIDINE RESIDUES OF S-PEPTIDE AND S-PROTEIN AND KINETICS OF H-1-H-2 EXCHANGE OF RIBONUCLEASE-A
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DOI：
10.1111/j.1432-1033.1977.tb11966.x
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发表时间：
1977-01-01
期刊：
影响因子：
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通讯作者：
TEH, JS
中科院分区：
文献类型：
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作者：
BRADBURY, JH;CROMPTON, MW;TEH, JS
100 MHz 的 1H NMR 光谱用于确定 35°C 2H2O 中 RNase-A 的 H-2 组氨酸共振的 1H-2H 交换的一级速率常数。 C 和 pH 计读数为 7、9、10 和 10.5。长时间暴露在 35°C 的 2H2O 中。 C和pH计读数11导致RNase-A不可逆变性。 pH 7 和 9 时的速率常数与 J. Ohe、H. Matsuo、F. Sakiyama 和 K. Narita 在 1H-3H 交换实验中获得的速率常数相当吻合。总结了不同作者获得的速率数据，并讨论了数据之间不一致的原因。随着 pH 值增加到 10.5，His-48 交换的一阶速率常数在 pH 9 时从接近零（由于其难以接近溶剂）迅速增加。在相同的 pH 范围内，His-119 的相应值显示出下降，而 His-12 的相应值则略有上升。这些变化归因于 RNase-A 铰链区的构象变化（可能是由于 Tyr-25 的滴定），这使得 His-48 能够接触到溶剂。 S-蛋白和S-肽以及在组氨酸残基的C-2位置部分氘化的材料的1H NMR谱证实了RNase-A的组氨酸共振的重新分配。跟踪 S 肽组氨酸 12 的 C-2 和 C-4 质子的化学位移作为 pH 的函数，得到 6.75 的 pK'' 值。 S 蛋白的三个 C-2 组氨酸共振的重新分配通过部分氘化研究得到证实。滴定His-48、His-105和His-119的H-2共振峰的pK''值分别为5.3、6.5和6.0。 S-蛋白对酸的稳定性不如 RNase-A，因为前者（而非后者）在 pH 3 和 26°C 下显示出可逆变性的证据。 C. S-蛋白中的 His-48 滴定正常，并且具有比 RNase-A 中更低的 pK，可能是因为缺少 Asp-14，而 Asp-14 在 RNase-A 中与 His-48 形成 H 键，导致其在 pH 值低于 9 时无法与溶剂接触。
1H NMR spectroscopy at 100 MHz was used to determine the 1st-order rate constants for the 1H-2H exchange of the H-2 histidine resonances of RNase-A in 2H2O at 35.degree. C and pH meter readings of 7, 9, 10 and 10.5. Prolonged exposure in 2H2O at 35.degree. C and pH meter reading 11 caused irreversible denaturation of RNase-A. The rate constants at pH 7 and 9 agreed reasonably well with those obtained in 1H-3H exchange experiments by J. Ohe, H. Matsuo, F. Sakiyama and K. Narita. The rate data obtained by various authors are summarized and the reasons for the poor agreement between the data are discussed. The 1st-order rate constant for the exchange of His-48 increases rapidly from near zero at pH 9 (due to its inaccessibility to solvent) with increase of pH to 10.5. The corresponding values for His-119 show a decrease and those for His-12 a small increase over the same pH range. These changes are attributed to a conformational change in the hinge region of RNase-A (probably due to the titration of Tyr-25) which allows His-48 to become accessible to solvent. 1H NMR spectra of S-protein and S-peptide, and of material partially deuterated at the C-2 positions of the histidine residues confirm the reassignment of the histidine resonances of RNase-A. The chemical shifts of the C-2 and C-4 protons of histidine-12 of S-peptide are followed as a function of pH and a pK'' value of 6.75 is obtained. The reassignment of the three C-2 histidine resonances of S-protein is confirmed by partial deuteration studies. The pK'' values obtained from titration of the H-2 resonances of His-48, His-105 and His-119 are 5.3, 6.5 and 6.0, respectively. The S-protein is less stable to acid than RNase-A since the former, but not the latter, shows evidence of reversible denaturation at pH 3 and 26.degree. C. His-48 in S-protein titrates normally and has a lower pK than in RNase-A probably because of the absence of Asp-14, which in RNase-A forms a H-bond with His-48 and causes it to be inaccessible to solvent, at pH values below 9.