Righetti P G, Bossi A, Gelfi C
University of Verona, Department of Agricultural and Industrial Biotechnologies, Italy.
J Capillary Electrophor. 1997 Mar-Apr;4(2):47-59.
The present review offers a new look at capillary isoelectric focusing (cIEF) by centering on the most troublesome aspects of the technique, namely: 1) how to modulate the slope of the pH gradient, for increasing resolution (equivalent to pH gradient engineering, as easily available in immobilized pH gradients); and 2) how to keep proteins in solution at (and in the proximity of) the pl value. A simple solution is offered in the first case: addition, to the standard 2-pH-units interval, of separators or spacers, i.e., of amphoteric molecules (either single or in combination) able to locally flatten the pH and increment resolution. Examples of the separation of fetal and glycated hemoglobins are provided. In the second case, a unique solubilization power (while maintaining full protein integrity and enzyme activity) is obtained if class I solubilizers are used. They consist of mixtures of sugars (e.g., sucrose and sorbitol) at ca. 1 M concentration, with zwitterions (up to 1 M) such as the class of nondetergent sulfobetaines, but also taurine and some of the Good's buffers (e.g., CAPS). In these solvents, the protein exists in a state of superhydration and its solubility is greatly augmented. The review ends with an excursus on the use of isoelectric buffers in zone electrophoretic separations. Such isoelectric buffers offer unique advantages: They permit very-high-voltage gradients (up to 1000 V/cm) and thus minimize analysis times (down to a few min in 30-35 cm long capillaries). This results in a marked increase in resolution, due to minimal diffusion-driven peak spreading. Such buffers are finding unique applications for generating peptide maps of tryptic digests of proteins and also in the analysis of oligonucleotides.
本综述通过聚焦该技术最棘手的方面，对毛细管等电聚焦（cIEF）进行了新的审视，即：1）如何调节pH梯度的斜率以提高分辨率（等同于固定化pH梯度中易于实现的pH梯度工程）；2）如何使蛋白质在其等电点值处（及附近）保持溶解状态。对于第一种情况，提供了一种简单的解决方案：在标准的2个pH单位间隔中加入分隔剂或间隔物，即能够局部平坦化pH并提高分辨率的两性分子（单一或组合使用）。文中给出了胎儿血红蛋白和糖化血红蛋白分离的示例。对于第二种情况，如果使用I类增溶剂，则可获得独特的增溶能力（同时保持蛋白质的完全完整性和酶活性）。它们由浓度约为1 M的糖类混合物（如蔗糖和山梨醇）与两性离子（浓度高达1 M）组成，这些两性离子包括非离子型磺基甜菜碱类，还有牛磺酸和一些Good缓冲液（如CAPS）。在这些溶剂中，蛋白质以超水化状态存在，其溶解度大大提高。综述最后附带讨论了等电缓冲液在区带电泳分离中的应用。此类等电缓冲液具有独特的优势：它们允许非常高的电压梯度（高达1000 V/cm），从而将分析时间减至最短（在30 - 35 cm长的毛细管中短至几分钟）。由于扩散驱动的峰展宽最小，这导致分辨率显著提高。此类缓冲液在生成蛋白质胰蛋白酶消化产物的肽图以及寡核苷酸分析中有着独特的应用。
