蛋白质的疏水相互作用色谱法V. 构象变化的定量评估
Hydrophobic interaction chromatography of proteins V. Quantitative assessment of conformational changes.
作者信息
Ueberbacher Rene, Haimer Emmerich, Hahn Rainer, Jungbauer Alois
机构信息
Department of Biotechnology, University of Natural Resources and Applied Life Sciences Vienna, Vienna, Austria.
出版信息
J Chromatogr A. 2008 Jul 11;1198-1199:154-63. doi: 10.1016/j.chroma.2008.05.062. Epub 2008 May 28.
Protein adsorption during hydrophobic interaction chromatography (HIC) may induce conformational changes. We analyzed conformational changes in three model proteins, bovine serum albumin (BSA), beta-lactoglobulin, and lysozyme by attenuated total reflectance Fourier transform infrared (ATR FT-IR) spectroscopy and pulse response experiments. Conformational changes occurred in the secondary structure of BSA, the tertiary structure of beta-lactoglobulin, and no changes occurred in lysozyme under the adsorption conditions investigated. Protein unfolding varied substantially among proteins, caused incomplete isocratic elution in HIC, and was confirmed by in situ assessments. Lower temperatures and binding capacities significantly reduced protein unfolding; the activation energy for unfolding ranged from 47 to 125 kJ/mol.
疏水作用色谱法（HIC）过程中的蛋白质吸附可能会引起构象变化。我们通过衰减全反射傅里叶变换红外光谱（ATR FT-IR）和脉冲响应实验分析了三种模型蛋白，即牛血清白蛋白（BSA）、β-乳球蛋白和溶菌酶的构象变化。在所研究的吸附条件下，BSA的二级结构、β-乳球蛋白的三级结构发生了构象变化，而溶菌酶未发生变化。蛋白质的去折叠在不同蛋白质之间有很大差异，导致HIC中不完全等度洗脱，并通过原位评估得到证实。较低的温度和结合容量显著降低了蛋白质的去折叠；去折叠的活化能范围为47至125 kJ/mol。
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