Kato Yoshio, Nakamura Koji, Kitamura Takashi, Moriyama Hiroyuki, Hasegawa Masazumi, Sasaki Hiroo
Nanyo Research Laboratory, Tosoh Corporation, Shinnanyo, Yamaguchi, Japan.
J Chromatogr A. 2002 Sep 20;971(1-2):143-9. doi: 10.1016/s0021-9673(02)01039-7.
We investigated protein separation by hydrophobic interaction chromatography (HIC) at low salt concentration on the supports of various hydrophobicities. Hydrophobic proteins could be successfully separated with more than 90% recovery by gradient elution of ammonium sulfate from 0.3-0.5 M to 0 in 50 mM phosphate buffer (pH 6.8) by using supports whose hydrophobicities were properly adjusted individually for each protein. Satisfactory results were also obtained by isocratic elution without ammonium sulfate and gradient elution of ethanol from 0 to 10%. HIC at low salt concentration was compatible with other modes of liquid chromatography like ion-exchange chromatography. On the other hand, it was not successful to separate hydrophilic proteins at low salt concentration. Recoveries of hydrophilic proteins decreased before they were retained enough as support hydrophobicity increased. Therefore, it is inevitable to use a higher concentration of salt, e.g., 1-2 M ammonium sulfate, on hydrophilic or moderately hydrophobic support in order to retain hydrophilic proteins without decrease in recovery.
我们研究了在低盐浓度下，利用各种疏水性载体通过疏水相互作用色谱法（HIC）进行蛋白质分离的情况。通过在50 mM磷酸盐缓冲液（pH 6.8）中，将硫酸铵从0.3 - 0.5 M梯度洗脱至0，使用针对每种蛋白质单独适当调整疏水性的载体，疏水性蛋白质能够以超过90%的回收率成功分离。在不使用硫酸铵的等度洗脱以及从0至10%乙醇梯度洗脱的情况下，也获得了满意的结果。低盐浓度下的HIC与离子交换色谱等其他液相色谱模式兼容。另一方面，在低盐浓度下分离亲水性蛋白质并不成功。随着载体疏水性增加，亲水性蛋白质在被充分保留之前回收率就降低了。因此，为了在不降低回收率的情况下保留亲水性蛋白质，在亲水性或中等疏水性载体上不可避免地要使用较高浓度的盐，例如1 - 2 M硫酸铵。
