Surface-mediated retention effects of subtilisin site-specific variants in cation-exchange chromatography.
Surface-mediated retention effects of subtilisin site-specific variants in cation-exchange chromatography.
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阳离子交换色谱中枯草杆菌蛋白酶位点特异性变体的表面介导保留效应。
DOI：
10.1016/s0021-9673(00)94793-9
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发表时间：
1988
期刊：
影响因子：
--
通讯作者：
Regnier,FE
中科院分区：
文献类型：
--
作者：
Chicz,RM;Regnier,FE
通过等度洗脱和一系列氯化钠浓度，在强阳离子交换柱上分离了野生型枯草杆菌菌素和几个特定部位的变异体。蛋白质表面初级序列的变化对枯草杆菌菌素的层析行为有明显的影响。这支持了这样的概念，即三维结构决定了哪些生物聚合物表面残基处于与固定相表面相互作用的位置。保留数据符合化学计量置换模型(SDM)。Lnk‘与ln1/[氯化钠]的关系图给出了蛋白质上与载体基质相互作用的离子基团平均数(Z)的值。将SDM用于变异体的层析保留，发现在低离子强度下蛋白质表面有一个不寻常的现象。SDM图通常提供lnk‘与L/[NaC l]之间的线性关系，斜率对应于Z数。这项研究揭示了两条在斜率和截距上不同的线，表明枯草杆菌菌素的Z数在洗脱液的中等离子强度时发生变化。这些结果归因于一些盐诱导的蛋白质表面事件，触发了结构的变化。对这种情况的层析检测反映了表面介导的事件和流动相离子强度之间的联系。
Wild-type subtilisin and several site-specific variants were resolved on a strong cation-exchange column by isocratic elution and a series of sodium chloride concentrations. Changes in primary sequence at the protein surface have an observable effect on the chromatographic behavior of subtilisin. This supports the concept that three-dimensional structure determines which biopolymer surface residues are in position to interact with the stationary phase surface.The retention data fit the stoichiometric displacement model (SDM) of retention. Plots of lnk′ vs.ln 1/[NaCl] yield values for the average number of ionic groups (Z) on the protein that interact with the support matrix. Application of the SDM to the chromatographic retention of the variants has uncovered an unusual phenomenon at the protein surface at low ionic strength.A SDM plot normally provides a linear relationship between lnk′ and l/[NaCl] with the slope corresponding to the Z number. This study revealed two lines differing in slope and intercept, indicating that the Z number of subtilisin changes at some intermediate ionic strength of the eluent. These results are attributed to some salt-induced protein surface event that triggers a change in structure. Chromatographic detection of this occurrence reflects the connection between the surface-mediated event and mobile phase ionic strength.