Fausnaugh J L, Regnier F E
J Chromatogr. 1986 May 30;359:131-46. doi: 10.1016/0021-9673(86)80068-1.
Hydrophobic interaction chromatography utilizes high salt concentrations 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 alpha-helical region starting with Residue 89. Retention was analyzed by plotting log k' versus the 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.
疏水相互作用色谱法利用高盐浓度的流动相来诱导弱疏水基质与天然蛋白质暴露的疏水氨基酸之间的相互作用。具有亲水性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，将从相关鸟类物种中分离出的溶菌酶在不断增加硫酸铵浓度的情况下在疏水相互作用柱上进行色谱分析。仅当溶菌酶与催化裂隙相对的表面发生氨基酸取代时，色谱保留才会出现偏差。该区域可能构成一个接触表面积，从第41位残基延伸至102位残基，从75位残基延伸至以第89位残基开始的α-螺旋区域。通过绘制log k'与硫酸铵的质量摩尔浓度的关系图来分析保留情况。每种鸟类溶菌酶的斜率均无显著偏差，表明接触表面积相似。然而，每种溶菌酶曲线的截距存在显著偏差，这可能反映了疏水相互作用的强度。随着溶菌酶变得更加疏水，截距增加。亲水性氨基酸取代对保留的影响与疏水性氨基酸取代一样大。溶菌酶与柱表面之间接触区域内组氨酸残基的电离状态也会影响保留。不带电荷的组氨酸残基会增加保留，而带电荷的组氨酸残基则会导致保留降低。氨基酸取代似乎并未影响疏水接触表面积的大小，而是影响了疏水相互作用的强度。盐组成对蛋白质保留的影响表明，除表面张力外的其他因素也可能影响保留。这些因素似乎包括蛋白质水化以及蛋白质与盐离子之间的特异性相互作用。其中，后者可能会也可能不会导致蛋白质结构的改变。发现盐组成的影响程度取决于蛋白质。
