SOLUTE AND MOBILE PHASE CONTRIBUTIONS TO RETENTION IN HYDROPHOBIC INTERACTION CHROMATOGRAPHY OF PROTEINS
SOLUTE AND MOBILE PHASE CONTRIBUTIONS TO RETENTION IN HYDROPHOBIC INTERACTION CHROMATOGRAPHY OF PROTEINS
复制标题
DOI：
10.1016/0021-9673(86)80068-1
复制
发表时间：
1986-05-30
期刊：
影响因子：
--
通讯作者：
REGNIER, FE
中科院分区：
文献类型：
--
作者：
FAUSNAUGH, JL;REGNIER, FE
疏水相互作用色谱法利用高盐浓度的移动的相来诱导弱疏水基质与天然蛋白质的暴露的疏水氨基酸之间的相互作用。具有亲水外部的蛋白质在疏水相互作用柱上的保留时间比具有更疏水外部的蛋白质短。为了研究氨基酸取代对蛋白质保留的影响，在增加硫酸铵浓度的疏水相互作用柱上对从相关鸟类中分离的溶菌酶进行色谱分析。只有当催化裂缝对面的溶菌酶表面发生氨基酸取代时，色谱保留才会发生偏离。该区域可以构成接触表面区域，并且从残基41延伸到102，从残基75延伸到从残基89开始的α-螺旋区域。通过将logk′与硫酸铵的摩尔浓度作图来分析保留。每种鸟溶菌酶的斜率没有显著偏离，表明接触表面积相似。然而，在每个溶菌酶线的截距中存在显著偏差，这可能反映了疏水相互作用的强度。随着溶菌酶的疏水性增加，截留量增加。亲水性氨基酸取代对保留的影响不亚于疏水性氨基酸取代。组氨酸残基在溶菌酶和柱表面之间的接触区域内的电离状态也影响保留。不带电荷的组氨酸残基增加了保留，而带电荷的组氨酸残基则减少了保留。氨基酸取代似乎不影响疏水接触表面积的大小，而是影响疏水相互作用的强度。盐组成对蛋白质保留的影响表明，除表面张力外，其他因素也会影响保留。这些因素似乎包括蛋白质水合作用和蛋白质与盐离子之间的特定相互作用。其中，后者可能会或可能不会导致蛋白质结构的改变。盐组合物的效果的大小被认为是依赖于蛋白质。
Hydrophobic interaction chromatography utilizes high salt concentration mobile phases to induce an interaction between a weakly hydrophobic matrix and exposed hydrophobic amino acids of a native protein. Proteins with a hydrophilic exterior have shorter retention times on a hydrophobic interaction column than do proteins with more hydrophobic exteriors. To examine the effect of amino acid substitutions on protein retention, lysozyme isolated from related bird species was chromatographed on a hydrophobic interaction column at increasing ammonium sulfate concentrations. Chromatographic retention deviated only when amino acid substitutions occurred on the surface of lysozyme opposite the catalytic cleft. This area may constitute a contact surface area and extends from Residue 41 to 102 and from 75 to the α-helical region starting with Residue 89. Retention was analyzed by plotting logk′versusthe molal concentration of ammonium sulfate. The slope did not deviate significantly for each of the bird lysozymes, indicating a similar contact surface area. However, there was significant deviation in the intercept of each of the lysozyme lines, which probably reflects the strength of the hydrophobic interaction. The intercept increased as the lysozyme became more hydrophobic. Hydrophilic amino acid substitutions affected retention as much as hydrophobic ones. The ionization state of histidine residues within the contact area between lysozyme and the column surface also influenced retention. An uncharged histidine residue increased retention, while a decrease in retention was seen with a charged histidine residue. The amino acid substitutions did not appear to affect the size of the hydrophobic contact surface area, but rather the strength of the hydrophobic interaction. The effect of salt composition on protein retention indicated that factors other than surface tension could influence retention. These factors appear to include protein hydration and specific interactions between the protein and the salt ions. Of these, the latter may or may not result in an alteration in protein structure. The magnitude of the effect of salt composition was found to be dependent upon the protein.