Hydrogen exchange kinetics of core peptide protons in Streptomyces subtilisin inhibitor.
Hydrogen exchange kinetics of core peptide protons in Streptomyces subtilisin inhibitor.
复制标题
链霉菌枯草杆菌蛋白酶抑制剂中核心肽质子的氢交换动力学。
DOI：
10.1021/bi00333a025
复制
发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Woodward,CK
中科院分区：
文献类型：
--
作者：
Akasaka,K;Inoue,T;Hatano,H;Woodward,CK
材料和方法粗品冻干SSI由大坂府立大学的Murao教授提供，通过Sato & Murao（1973）的方法进一步纯化。在氢交换测量之前，将纯化的冻干SSI溶解在2 H2O中，用NaO 2 H调节至pH 9，并在45 ℃下孵育12-18小时以使快速交换的质子氘化。将溶液的等分试样与等体积的氘代缓冲液混合，得到36-75 mg/mL SSI、0.1 M NaCl和0.1 M缓冲液的最终浓度。在交换实验之前和之后检查样品的pH。所用缓冲液为pH 7-8和pH 11的磷酸钠、pH 8-10的硼酸钠和pH 10- 11的碳酸钠。NMR光谱在尼科莱NT光谱仪上使用5 mm直径的管在300 MHz下测量。施加具有1秒的总重复的45采样脉冲，或具有2秒的总重复的90采样脉冲。在100-1000次扫描中累积自由感应衰减信号。探针温度调节到±1 ℃，通过羟基质子的化学位移监测，
Materials and MethodsCrude lyophilized SSI, kindly supplied by Professor Murao of Osaka Prefectural University, was further purified by the method of Sato & Murao (1973). Prior to the hydrogen exchange measurements, purified, lyophilized SSI was dis-solved in 2H20, adjusted to pH 9 with Na02H, and incubated at 45 C for 12-18 h to deuterate the rapidly exchanging protons. An aliquot of the solution was mixed with an equal volume of deuterated buffer to give a final concentration of 36-75 mg/mL SSI, 0.1 M NaCl, and 0.1 M buffer. The pH of the sample was checked before and after the exchange experiment. Buffers used were sodium phosphate at pH 7-8 and pH 11, sodium borate at pH 8-10, and sodium carbonate at pH 10-11.The NMR spectra were measured on a Nicolet NT spectrometer operating at 300 MHz using 5-mm-diameter tubes. A 45 sampling pulse with a total repetition of 1 s, or a 90 pulse with a total repetition of 2 s, was applied. The free induction decay signals were accumulated over 100-1000 scans. The probe temperature, regulated to±1 C, was monitored by the chemical shiftof the hydroxy proton of