Abstract: The heparin affinity chromatography is widely applied for purification of proteins due to its high specificity and ease of operation. The heparin affinity chromatography medium was prepared through Schiff base method using heparin as the ligand, which was based on the macroporous polyacrylate microspheres. Firstly, the epoxy groups in the macroporous microspheres were hydrolyzed to be o-hydroxy through 0.5 mol/L H2SO4 aqueous solution. Secondly, the o-hydroxy was further oxidized to be aldehyde groups. Finally, the heparin was immobilized in the macroporous microsphere through the reaction between aldehyde groups and amino groups. The effects of operating conditions on the coupling reaction were investigated and optimized including the concentration of heparin, pH, buffer concentration, and reaction time. The effect of the reaction factor on the adsorption of proteins was evaluated using lysozyme as model protein. The optimal reaction conditions were found and the static binding capacity of the model proteins reached 40.3 mg/mL. This value was about 36% higher than that of the commercial GP-heparin. The protein recovery in this medium reached 95% with 1.0 mol/L NaCl as elution solvent. The morphology of the microspheres was observed by scanning electron microscopy. The result showed that the throughput pores were maintained in the affinity medium. The dynamic binding capacity of lysozyme on the affinity support was determined under different flow rates (31.8~318 cm/h). The result indicated the capacity at 318 cm/h decreased by about 12% in comparison with that at 31.8 cm/h. The dynamic binding capacity remained 81% of the initial value after 10 cycles. The synthesized affinity medium was used to isolate lactoferrin from mixtures. The results showed that it had a high separation efficiency.

Key words: affinity chromatography, heparin, macroporous chromatography medium, coupling method, Schiff base method

摘要： 肝素亲和层析介质因其专一性好、操作条件温和等特点，广泛应用于蛋白分离纯化，本工作以大孔聚丙烯酸酯微球为基质，肝素为配基，利用席夫碱法制备了肝素亲和层析介质。配基偶联经过三步完成，首先将大孔聚丙烯酸酯微球表面环氧基团通过0.5 mol/L H2SO4水解为邻羟基，然后将邻羟基氧化为醛基，最后利用醛基与肝素分子的胺基反应将肝素分子固定于微球表面。以溶菌酶为模型蛋白，主要考察了肝素偶联反应的各因素对蛋白结合容量的影响规律，包括肝素浓度、缓冲液pH及浓度、反应时间等，建立了最优偶联肝素配基的方法。所得亲和介质静态结合容量可达40.3 mg/mL，比商品GP-肝素介质高约36%，经1.0 mol/L的氯化钠洗脱，其蛋白回收率达到95%。通过扫描电子显微镜表征微球表面形貌，观察到偶联肝素后的微球仍能保持其大孔结构。考察了该类亲和介质在不同操作流速(31.8~318 cm/h)下的动态结合容量，发现操作流速提高10倍后介质结合容量仅下降12%。经10次重复使用后，动态结合容量仍可保持初始容量的81%。用于混合蛋白模型中分离乳铁蛋白，结果表明具有良好的分离效果。

关键词: 亲和层析, 肝素, 大孔层析介质, 偶联方法, 席夫碱法