Ozyurt Sinem, Kirdar Betül, Ulgen Kutlu O
Department of Chemical Engineering, Bogaziçi University, Bebek, Istanbul, Turkey.
J Chromatogr A. 2002 Jan 25;944(1-2):203-10. doi: 10.1016/s0021-9673(01)01102-5.
In the present work, a new method of purification for antithrombin was developed using an expanded bed chromatography technique. Milk fat was removed by centrifugation and caseins were precipitated selectively by addition of zinc chloride. Crude skim milk was then directly fed to an expanded bed column containing the ion-exchange matrix. The use of a cation-exchanger (P-11) resulted in 100% adsorption and 13% recovery whereas the use of an anion-exchanger (DE-52) resulted in 100% adsorption and 84% recovery and up to five-fold purification of antithrombin. The buffer, 25 mM Tris-HCl pH 8.0; the eluting agent, 2 M (NH4)2SO4; and 100% expansion of settled bed were determined to be the optimum conditions for the purification of antithrombin by ion-exchange expanded bed chromatography. A comparison of column diameters revealed that the elution yields increase by two-fold while the column diameter increases from 1 to 2.5 cm. However, antithrombin III was concentrated to a higher degree by using the column with an internal diameter of 1 cm.
在本研究中，开发了一种使用扩张床色谱技术纯化抗凝血酶的新方法。通过离心去除乳脂肪，并通过添加氯化锌选择性沉淀酪蛋白。然后将粗脱脂乳直接进料到含有离子交换基质的扩张床柱中。使用阳离子交换剂（P-11）时吸附率为100%，回收率为13%；而使用阴离子交换剂（DE-52）时吸附率为100%，回收率为84%，抗凝血酶的纯化倍数高达5倍。确定缓冲液为25 mM Tris-HCl pH 8.0；洗脱剂为2 M (NH4)2SO4；固定床100%的膨胀率为通过离子交换扩张床色谱法纯化抗凝血酶的最佳条件。柱直径的比较表明，当柱直径从1 cm增加到2.5 cm时，洗脱产率提高两倍。然而，使用内径为1 cm的柱子时，抗凝血酶III的浓缩程度更高。
