Sullivan R C, Shing Y W, D'Amore P A, Klagsbrun M
J Chromatogr. 1983 Aug 26;266:301-11. doi: 10.1016/s0021-9673(01)90904-5.
Size-exclusion and ion-exchange high-performance liquid chromatography were used to purify biologically active growth factors as measured by the ability of the factors to stimulate DNA synthesis in 3T3 cells. Chromatography was performed in aqueous buffer and at neutral pH to avoid possible inactivation of biological activity. The growth factors analyzed were chondrosarcoma growth factor (CHSA-GF), human milk growth factor (HMGF), retinal-derived growth factor (RDGF) and mouse epidermal growth factor (EGF). CHSA-GF, HMGF, and RDGF were eluted from TSK 2000 columns as well-defined peaks of activity with molecular weights of 12,000-15,000, 5000-6000, and 16,000-18,000, respectively. EGF was found to have an abnormally low molecular weight after chromatography on TSK 2000. However, incorporation of guanidine . HCl into the TSK column resulted in the elution of EGF at its known molecular weight of ca. 6000. Anion-exchange high-performance liquid chromatography on AX 300 was used for the purification of HMGF and RDGF, and cation-exchange high-performance liquid chromatography on CM 300 was used for the purification of CHSA-GF. The results show that size-exclusion and ion-exchange chromatography can be used without organic solvents or extremes in pH to purify a number of different growth factors successfully with retention of biological activity.
尺寸排阻和离子交换高效液相色谱法被用于纯化生物活性生长因子，其通过这些因子刺激3T3细胞中DNA合成的能力来衡量。色谱分析在水性缓冲液中且在中性pH下进行，以避免生物活性可能的失活。所分析的生长因子有软骨肉瘤生长因子（CHSA-GF）、人乳生长因子（HMGF）、视网膜衍生生长因子（RDGF）和小鼠表皮生长因子（EGF）。CHSA-GF、HMGF和RDGF从TSK 2000柱上以定义明确的活性峰形式洗脱，分子量分别为12,000 - 15,000、5000 - 6000和16,000 - 18,000。在TSK 2000柱上进行色谱分析后，发现EGF的分子量异常低。然而，在TSK柱中加入盐酸胍后，EGF以其已知分子量约6000被洗脱。AX 300上的阴离子交换高效液相色谱法用于纯化HMGF和RDGF，CM 300上的阳离子交换高效液相色谱法用于纯化CHSA-GF。结果表明，尺寸排阻和离子交换色谱法可以在不使用有机溶剂或极端pH条件下成功纯化多种不同的生长因子，并保留其生物活性。
