Abstract: Direct separation and purification of hemoglobin from fresh bovine hemolysate were studied with expanded bed adsorption. Ionic strength, pH and temperature had great influence on the formation of methemoglobin in the process. The purity and recovery of the product were also influenced by these factors. The conditions for adsorption and elution were optimized. The best performance was obtained by using 10mmol•L^-1 phosphate buffer solution, pH6.6. Eluting at pH 7.2 was carried out at low ionic strength and the final recovery of hemoglobin was more than 80%. A low temperature of 4℃ in the process was necessary for prevention of methemoglobin formation. Under optimized conditions, natural bovine hemoglobin of electrophoretical purity could be obtained by only one step of operation, and the dynamic adsorption capacity was between 70—75mg•ml^-1. HPLC result suggested that the purity of the product was about 99%.Methemoglobin was kept as low as 5.4%. Compared with the traditional process of membrane filtration followed by chromatography, the expanded bed adsorption has the advantages of short processing time, high recovery, and high product activity. The method was simple, efficient and potential scale-up to commercial production.

Key words:

膨胀床吸附, 纯化, 牛血红蛋白, Streamline SP

摘要： 探索了将膨胀床吸附层析应用于动物血液蛋白质的提取，尝试了直接从牛血红细胞破碎液中纯化血红蛋白.对该过程采用的吸附介质及pH值、离子强度、温度、洗脱条件等因素对高铁血红蛋白生成、产品纯度以及回收率等的影响进行探索.选择了最佳吸附介质Streamline SP，确定了优化的吸附条件为：吸附过程选用离子强度为10mmol•L^-1的磷酸盐缓冲系统，进料线流速3.3cm•min^-1，进料pH值6.6，改变缓冲液pH值为7.2进行洗脱，操作温度4℃.该技术省去了离心去除碎片步骤，有效地控制了纯化过程中无载氧能力的高铁血红蛋白的生成，动态吸附容量达到70～75mg•ml^-1，只经一步操作产品纯度达电泳纯，高铁血红蛋白含量仅为5.4％.与现有的膜过滤-层析纯化方法相比，膨胀床吸附法操作时间短，提取收率高，产品活性损失小，是一种简捷、高效、适合工业放大的天然血红蛋白制备方法.

关键词:

膨胀床吸附, 纯化, 牛血红蛋白, Streamline SP