Mant C T, Zhou N E, Hodges R S
Department of Biochemistry, University of Alberta, Edmonton, Canada.
J Chromatogr. 1989 Aug 4;476:363-75. doi: 10.1016/s0021-9673(01)93882-8.
The use of amino acid retention or hydrophobicity coefficients for the prediction of peptide retention time behaviour on hydrophobic stationary phases is based on the premise that amino acid composition is the major factor affecting peptide retention in reversed-phase chromatography. Although this assumption holds up well enough for small peptides (up to ca. 15 residues), it is now recognized that polypeptide chain length must be taken into account when attempting to equate retention time behaviour of larger peptides and proteins with their overall hydrophobicity. In the present study, we have examined the reversed-phase retention behaviour of 19 proteins of known sequence on stationary phases of varying hydrophobicity and ligand density. From the observed protein retention behaviour on C4, C8 and C18 stationary phases under gradient elution conditions, we have been able to correlate the observed retention times of proteins ranging in molecular weight from 3500 to 32,000 dalton and in chain length from 30 to 300 residues with their overall hydrophobicity (based on retention parameters derived from small peptides) and the number of residues in the polypeptide chain. The retention behaviour of the proteins on the C4, C8 and C18 columns was also compared to that obtained on supports containing lower ligand densities (phenyl ligands). The maintenance of native or partially folded protein conformation on the phenyl columns, resulting in lower retention times than would be expected for fully denatured proteins, underlined the importance of efficient protein denaturation for satisfactory correlation of protein retention times with protein hydrophobicity. In addition, the effectiveness of increasing temperature and/or ligand density of the stationary phase in denaturing proteins was also demonstrated.
使用氨基酸保留系数或疏水性系数来预测肽在疏水固定相上的保留时间行为，其前提是氨基酸组成是影响反相色谱中肽保留的主要因素。尽管这一假设对于小肽（最多约15个残基）来说足够成立，但现在人们认识到，在试图将较大肽和蛋白质的保留时间行为与其整体疏水性等同起来时，必须考虑多肽链长度。在本研究中，我们检测了19种已知序列的蛋白质在不同疏水性和配体密度的固定相上的反相保留行为。根据在梯度洗脱条件下观察到的蛋白质在C4、C8和C18固定相上的保留行为，我们能够将分子量在3500至32000道尔顿、链长在30至300个残基范围内的蛋白质的观察保留时间与其整体疏水性（基于从小肽得出的保留参数）和多肽链中的残基数相关联。还将蛋白质在C4、C8和C18柱上的保留行为与在含较低配体密度（苯基配体）的载体上获得的保留行为进行了比较。在苯基柱上天然或部分折叠的蛋白质构象得以维持，导致保留时间比完全变性的蛋白质预期的要短，这突出了有效蛋白质变性对于使蛋白质保留时间与蛋白质疏水性得到满意关联的重要性。此外，还证明了提高温度和/或固定相配体密度在使蛋白质变性方面的有效性。
