Purcell A W, Zhao G L, Aguilar M I, Hearn M T
Department of Biochemistry and Molecular Biology, Centre for Bioprocess Technology, Monash University, Clayton, Vic., Australia.
J Chromatogr A. 1999 Aug 6;852(1):43-57. doi: 10.1016/s0021-9673(99)00440-9.
The isocratic and gradient elution behaviour of beta-endorphin and glucagon, two polypeptides known to exist in amphipathic alpha-helical conformations in lipophilic environments, have been examined under reversed-phase high-performance liquid chromatographic (RP-HPLC) conditions with low pH, aquo-acetonitrile mobile phases. The effects of changes in the volume fraction, psi, of the organic solvent modifier and temperature, T, on the magnitudes of the S and log k(o) values of these two polypeptides, obtained from the plots of logarithmic capacity factor (log k') vs. psi using isocratic elution conditions have been determined. These data have then been compared to the corresponding S and log k(o) values, obtained from the plots of logarithmic median capacity factor (log k) versus the median volume fraction of the organic solvent modifier (psi) derived from the linear gradient elution data, using the same n-butyl silica sorbent and related aquo-acetonitrile mobile phase conditions. As apparent from these studies, substantial differences occur in the temperature-dependent trends and magnitudes of the corresponding S and S values, or the log k(o) and log k(o) values, when these parameters are derived from experimental data acquired by these two different elution methods. Moreover, when gradient elution data for beta-endorphin and glucagon are utilised, the extrapolated values of the intercept and slope of the plots of log k vs. 1/T (corresponding to an apparent change in the median enthalpy of association, deltaH(o)assoc, or an apparent change in the median entropy of association, deltaS(o)assoc) substantially deviated from the values obtained for the thermodynamic parameters, deltaH(o)assoc and deltaS(o)assoc, derived from the log k' vs. 1/T plots using the corresponding isocratic data. These findings thus have important implications for biophysical and thermodynamic investigations when gradient elution data are employed to assess the molecular basis of the interaction of polypeptides with non-polar ligates.
β-内啡肽和胰高血糖素这两种已知在亲脂环境中以两亲性α-螺旋构象存在的多肽，在低pH值的水-乙腈流动相的反相高效液相色谱（RP-HPLC）条件下，对其等度洗脱和梯度洗脱行为进行了研究。通过等度洗脱条件下对数容量因子（log k'）对ψ的作图，确定了有机溶剂改性剂的体积分数ψ和温度T的变化对这两种多肽的S和log k(o)值大小的影响。然后，使用相同的正丁基硅胶吸附剂和相关的水-乙腈流动相条件，将这些数据与从线性梯度洗脱数据得出的对数中值容量因子（log k）对有机溶剂改性剂的中值体积分数（ψ）的作图中获得的相应S和log k(o)值进行比较。从这些研究中可以明显看出，当这些参数从通过这两种不同洗脱方法获得的实验数据中得出时，相应的S和S值或log k(o)和log k(o)值的温度依赖性趋势和大小存在显著差异。此外，当利用β-内啡肽和胰高血糖素的梯度洗脱数据时，log k对1/T作图的截距和斜率的外推值（对应于缔合中值焓的表观变化，ΔH(o)assoc，或缔合中值熵的表观变化，ΔS(o)assoc）与使用相应等度数据从log k'对1/T作图得出的热力学参数ΔH(o)assoc和ΔS(o)assoc的值有很大偏差。因此，当使用梯度洗脱数据来评估多肽与非极性连接体相互作用的分子基础时，这些发现对生物物理和热力学研究具有重要意义。
