Watanabe E, Tsoka S, Asenjo J A
Biochemical Engineering Laboratory, University of Reading, United Kingdom.
Ann N Y Acad Sci. 1994 May 2;721:348-64. doi: 10.1111/j.1749-6632.1994.tb47407.x.
The charge characteristics of four proteins (conalbumin, ovalbumin, apotransferrin, and soybean trypsin inhibitor) were determined over a range of pH using an electrophoretic technique (titration curve). HPLC anion-exchange chromatography for each of the proteins was performed to investigate whether electrophoretic and chromatographic results could be correlated. It was found that charge density, estimated from the electrophoretic titration curves (as net charge divided by the protein molecular weight) in most cases correlates extremely well the retention of the protein in terms of time needed for the protein to be eluted from the column. This correlation was far better than that of net charge or that of surface charge (net charge/surface area). The performance of anion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration using preparative columns on an FPLC were compared. The ability of each method to separate the components of the mixture of the four proteins was assessed by calculating the resolution of the chromatographic results obtained for each pair of proteins. Using the concept of the deviation factor (DF) between individual properties of pairs of proteins (charge density, molecular weight, hydrophobicity), the separation efficiency factor (eta s) could be calculated. Resolution results showed that anion exchange was the best technique for separating the proteins followed by hydrophobic interaction chromatography and gel filtration. Calculations for efficiency, eta s, confirmed the superiority of anion-exchange chromatography as a separation method over gel filtration. For hydrophobic interaction chromatography, it was not possible to find a suitable parameter to express DF and, consequently, eta s. An appropriate way to express the hydrophobicity of a protein, as a physicochemical property, that can be used in the expression of DF should be investigated.
采用电泳技术（滴定曲线）在一定pH范围内测定了四种蛋白质（伴清蛋白、卵清蛋白、脱铁转铁蛋白和大豆胰蛋白酶抑制剂）的电荷特性。对每种蛋白质进行了高效液相色谱阴离子交换色谱分析，以研究电泳结果与色谱结果是否相关。结果发现，根据电泳滴定曲线估算的电荷密度（净电荷除以蛋白质分子量）在大多数情况下与蛋白质在柱上洗脱所需时间的保留情况具有极好的相关性。这种相关性远优于净电荷或表面电荷（净电荷/表面积）的相关性。比较了在FPLC上使用制备柱进行阴离子交换色谱、疏水相互作用色谱和凝胶过滤的性能。通过计算每种蛋白质对的色谱结果的分辨率，评估了每种方法分离四种蛋白质混合物成分的能力。利用蛋白质对的个体性质（电荷密度、分子量、疏水性）之间的偏差因子（DF）概念，可以计算分离效率因子（ηs）。分辨率结果表明，阴离子交换是分离蛋白质的最佳技术，其次是疏水相互作用色谱和凝胶过滤。效率计算ηs证实了阴离子交换色谱作为一种分离方法优于凝胶过滤。对于疏水相互作用色谱，无法找到合适的参数来表示DF，因此也无法表示ηs。应该研究一种合适的方法来表达蛋白质的疏水性，作为一种物理化学性质，可用于DF的表达。
