一种安全高效的血红蛋白纯化方法

目的:建立一种适用于大量制备的,安全、高效的血红蛋白纯化方法。方法: 将压积红细胞装入透析袋,以含有还原剂的Tris缓冲液透析破碎,破碎的上清经两级硫酸铵沉淀后透析至上样缓冲体系,离心后取上清即得血红蛋白提取液;红细胞提取液通过阴离子交换柱层析进一步分离,计算回收率。纯化产物浓缩后以SDS-PAGE及HPLC鉴定纯度,进行紫外-可见光谱扫描并以ABL800血气分析仪分析血气指标,以鲎试剂测定内毒素含量,以磷测定法测定脂质含量。结果: 血红蛋白提取液中脂质去除率98%,容易通过0.45μm滤膜;经阴离子交换层析纯化的血红蛋白经SDS-PAGE(银染法)及WB分析没有杂蛋白条带,HPLC分析纯度>99%、总回收率>85%;内毒素含量<2 EU,高铁血红蛋白含量<5%。结论: 该血红蛋白纯化方法安全高效、成本低廉、易于放大生产,具有较好的应用前景。

An Efficient and Safe Method for Hemoglobin Purification

Objective: To build up a hemoglobin purification method which could meet the needs of safety, efficiency and scale amplification. Methods: Packed red blood cells were lysed by dialyzing against 10 mmol/L Tris-HCl (pH 8.0) containing 0.15% (M/V) ascorbic acid at 4℃ for 3 h with the buffer changed once an hour, and then centrifuged at 10 000 g for 10 min (4℃). The supernatant was treated with ammonium sulfate by the two-step salting-out operation to the saturation of 20% and 46% (M/V, calibrated at 0℃) subsequently, and then the precipitated protein was re-suspended using the equalizing buffer (25 mmol/L Tris, 10 mmol/L NaCl pH 8.5) with the equal volume to that of the packed red blood cells. After dialyzing against the equalizing buffer for at 4℃ for 3 h with the same changing interval, the protein solution was diluted with the equalizing buffer by the ration of 1∶2 (V/V) and centrifuged at 12 000 g for 20 min (4℃). The supernatant after centrifugation, namely the rough hemoglobin extract, was loaded to an 50 mm*200 mm (d*h) Q sapharose FF anion exchange chromatography column pre-equalized with the equalizing buffer and then hemoglobin was eluted using 25 mmol/L Tris, 80 mmol/L NaCl pH 8.5. The purity of samples from different processed was analyzed by SDS-PAGE and HPLC, and the oxygen-binding status was characterized by the UV-Vis spectrum scanning and blood-gas indexes, which were provided by the Thermo Multiscan Spectrum and ABL800 Flex Blood-gas Analyzer respectively. Lipoprotein concentration was tested by the phosphorus determination method and LPS was determined using the stachypleus amebocyte lysate with the sensitivity of 0.3 EU/ml, 0.1 EU/ml and 0.03 EU/ml. Results more than 98% lipids were removed in the rough hemoglobin extracts compared with hemoglobin extracts prepared by the conventional method, and this rough hemoglobin extracts could easily go through the 0.45 μm cellulose nitrate membrane. In the following anion exchange chromatography process, it could be observed that column contaminants were efficiently removed by the two-step ammonium sulfate precipitation. No other protein but hemoglobin could be detected in the hemoglobin purified by anion exchange chromatography through SDS-PAGE (silver staining) and WB, and HPLC analysis indicated a purity of above 99%. The total recovery of hemoglobin in the anion exchange chromatography was above 85%, and most hemoglobin molecules were in the ferrous oxyhemoglobin status and methemoglobin was less than 5% in the purified hemoglobin revealed by UV-Vis spectrum scanning and blood-gas determination. LPS was less than 2EU/ml. Conclusion: The hemoglobin purification method introduced above could meet the need of safety, efficiency, cost control and amplification, and a good application could be expected in the future.