Jones Tara Tibbs, Fernandez Erik J
Department of Chemical Engineering, University of Virginia, 102 Engineers' Way, PO Box 400741, Charlottesville, Virginia 22904-4741, USA.
Biotechnol Bioeng. 2004 Aug 5;87(3):388-99. doi: 10.1002/bit.20123.
Interesting retention and selectivity changes have been noted for a number of proteins in hydrophobic interaction chromatography (HIC). In this study, we investigated the degree to which conformational changes may be responsible for selectivity changes of stable proteins. Hydrogen-deuterium isotope exchange detected by mass spectrometry was used to investigate changes in solvent accessibility during adsorption on HIC media. Lysozyme was determined to exhibit EX2 hydrogen exchange kinetics both in solution and adsorbed to Butyl Sepharose 4 Fast Flow and Phenyl Sepharose 6 Fast Flow high sub surfaces. A small, but significant, increase in solvent accessibility was observed upon adsorption. Similar approaches were used to analyze solvent accessibility of three stable proteins with melting temperatures above 50 degrees C exhibiting significant selectivity changes on Butyl Sepharose and Toyopearl Butyl 650M. While all three proteins (lysozyme, chymotrypsinogen A, and ovalbumin) exhibited enhanced exchange while adsorbed, no differences in solvent accessibility on the different adsorbents were observed. More detailed studies of lysozyme showed no significant changes in labeling prior or during elution. These results demonstrate that HIC surfaces examined here do not dramatically alter the structure of these stable proteins and that differences in conformation are not responsible for the selectivity changes observed. Thus, other factors such as different preferred binding orientations or variations between the media pore structure, size, and/or surface chemistry must be responsible.
在疏水相互作用色谱法（HIC）中，已注意到许多蛋白质的保留和选择性发生了有趣的变化。在本研究中，我们调查了构象变化对稳定蛋白质选择性变化的影响程度。通过质谱检测的氢-氘同位素交换用于研究在HIC介质上吸附过程中溶剂可及性的变化。已确定溶菌酶在溶液中以及吸附到丁基琼脂糖4 Fast Flow和苯基琼脂糖6 Fast Flow高取代表面时均表现出EX2氢交换动力学。吸附后观察到溶剂可及性有小幅但显著的增加。使用类似的方法分析了三种熔点高于50摄氏度的稳定蛋白质在丁基琼脂糖和Toyopearl丁基650M上的溶剂可及性，它们表现出显著的选择性变化。虽然所有三种蛋白质（溶菌酶、胰凝乳蛋白酶原A和卵清蛋白）在吸附时都表现出增强的交换，但在不同吸附剂上未观察到溶剂可及性的差异。对溶菌酶更详细的研究表明，在洗脱之前或洗脱过程中标记没有显著变化。这些结果表明，此处研究的HIC表面不会显著改变这些稳定蛋白质的结构，并且构象差异不是观察到的选择性变化的原因。因此，其他因素，如不同的优先结合取向或介质孔结构、大小和/或表面化学之间的差异，必定是造成这种情况的原因。
