中国生物工程杂志

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.