Jennissen Herbert P
Department of Physiological Chemistry, University of Duisberg-Essen, Germany.
Methods Mol Biol. 2005;305:81-99.
Hydrophobic interaction chromatography (HIC) is one of the basic purification procedures in the biosciences. However, because of its complexity, it has not gained the same foothold in the methodological repertoire of protein chemistry as has affinity chromatography or ion exchange chromatography. This is mainly a result of the lack of a general optimization procedure for the reversible adsorption and elution of a novel protein to be purified. Further problems arise from the fact that most commercial hydrophobic adsorbents are inadequate for an ideal performance in downstream processing procedures, because these media are too hydrophobic and elution of proteins in their native state is often impossible. Therefore, as in the 1970s, a bioscientist of today has to be capable of synthesizing a small library of hydrophobic gels from which he or she can then select and optimize the ideal matrix for their special needs. In addition, a general optimization method employing the critical hydrophobicity concept has now been devised that should allow the application of HIC methodology to many hitherto unpurified proteins. In this chapter, the reader is first introduced to the theoretical background (multivalence, negative cooperativity, adsorption hysteresis) of the binding of protein ligands to hydrophobic supports, so that they will be capable of independently adapting HIC to a novel protein. Then a simple nontoxic method is described for the synthesis of HIC-gel libraries consisting of a homologous series of uncharged alkyl-Sepharoses of three chain lengths (butyl, pentyl, and hexyl Sepharose) prepared with different degrees of separation. From this series a critical hydrophobicity gel can then be selected and employed for critical hydrophobicity HIC. A detailed example for the chromatography of human fibrinogen is given that has been employed as a one-step procedure for the purification of fibrinogen from human plasma.
疏水作用色谱法（HIC）是生物科学中的基本纯化方法之一。然而，由于其复杂性，它在蛋白质化学的方法库中并未像亲和色谱法或离子交换色谱法那样站稳脚跟。这主要是由于缺乏一种通用的优化程序来实现待纯化新蛋白质的可逆吸附和洗脱。进一步的问题源于这样一个事实，即大多数商业疏水吸附剂在下游加工过程中无法实现理想性能，因为这些介质过于疏水，通常无法洗脱天然状态的蛋白质。因此，就像在20世纪70年代一样，如今的生物科学家必须能够合成一个小型疏水凝胶库，然后从中选择并优化出满足其特殊需求的理想基质。此外，现在已经设计出一种采用临界疏水性概念的通用优化方法，该方法应能使HIC方法应用于许多迄今未纯化过的蛋白质。在本章中，首先向读者介绍蛋白质配体与疏水支持物结合的理论背景（多价性、负协同性、吸附滞后），以便他们能够独立地将HIC应用于新蛋白质。然后描述一种简单的无毒方法，用于合成由三种链长（丁基、戊基和己基琼脂糖）的同源系列不带电荷的烷基琼脂糖组成的HIC凝胶库，这些琼脂糖具有不同程度的分离度。然后可以从该系列中选择临界疏水性凝胶并用于临界疏水性HIC。给出了人纤维蛋白原色谱分析的详细示例，该方法已被用作从人血浆中纯化纤维蛋白原的一步法。
