用参数Z表征疏水色谱中脲变α-糜蛋白酶及其折叠中间体的分子构象变化  

Characterization of the molecular conformational changes of urea-denatured α-Chymotrypsin and its folded intermediate by parameter Z in high performance hydrophobic interaction chromatography

作　　者：耿信笃[1] 刘振岭[1] 柯从玉[1] 李建军[1] 

机构地区：[1]西北大学现代分离科学研究所/合成与天然功能分子化学教育部重点实验室,陕西西安710069

出　　处：《西北大学学报（自然科学版）》2007年第6期879-885,共7页Journal of Northwest University（Natural Science Edition）

基　　金：国家自然科学基金资助项目(39880003;20175916)

摘　　要：目的用参数Z表征疏水色谱中脲变α-糜蛋白酶（α-Chy）及其折叠中间体的分子构象变化。方法用疏水色谱法对不同脲变条件下的α-Chy进行分离，对收集的不同馏分用基质辅助激光解吸附离子化飞行时间质谱（HPHIE-MALDI-TOF-MS）进行检测，并用紫外和荧光光谱对其分子构象变化进行了验证。结果确认了脲变α-Chy中仅有一个稳定的折叠中间体存在，发现二者的z值均随脲浓度的增加而减小。在脲浓度范围为0～3．0mol·L^-1时，α-Chy折叠中间体的Z值远小于天然α-Chy的Z值，而脲浓度为4．0mol·L^-1和5．0mol·L^-1时，折叠中间体的Z值稍大于天然α-Chy的Z值。结论α-Chy折叠中间体的分子构象随脲浓度改变基本呈连续性变化。而天然α-Chy的分子构象随脲浓度呈不连续性变化。进一步发现α-Chy的Z值与其复性回收率之间存在着相似的变化规律。中间体的保留机理服从计量置换保留理论（SDT-R），且得到的参数Z可用来表征天然α-Chy和其折叠中间体的分子构象变化。Aim The parameter Z was used to characterize the c natured α-Chymotrypsin （α-Chy） and its intermediate. Methods hanges of molecular conformation for both urea-de- The denatured α-Chy by different concentrations of Urea was isolated by high-performance hydrophobic interaction chromatography （HPHIC）. The collected fractions were also indentified by matrix-assisted laser desorption inonization time of flight mass spectrometer （ MALDI-TOF- MS）. Results It was confirmed that there is only a stably folded intermediate of the urea-denatured α-Chywas formed,Their Z values obtained from the native and urea-denatured α-Chy were found to decrease with the increasing in urea concentration in the mobile phase employed. The folded intermediate has much less Z value than its native one as the urea concentration changing in the range of 0 ～ 3.0 mol · L^-1, while the former has little bigger than the latter one as urea concentration is from 4.0 mol· L^-1 to 5.0 mol· L^-1. Conclusion It was found that as urea concentration changing, the molecular conformation of the folded intermediate appears a continuous change, while that of its native one does a discontinuous change, and further found that the Z value and the bioactivity recovery of α-Chy tend to change with a same regularity. The retention mechanism of the intermediate was found to obey the stoichiometric displacement theory for retention （SDT-R） and the obtained parameter Z could be used to char- acterize the changes in the molecular conformation changes for both native α-Chy and its intermediate.

关 键 词：蛋白折叠 分子构象 疏水相互作用色谱 基质辅助激光解吸附离子化飞行时间质谱 (MALDI-TOF-MS) 计量置换 α-糜蛋白酶(α-Chy) 折叠中间体 

分 类 号：O658[理学—分析化学]