纯化工艺对牛血红蛋白携氧/释氧能力的影响研究  

Effect of purification process on oxygen carrying/releasing capacity of bovine hemoglobin

作　　者：秦东 李伟丹 盛铭 高道远 褚宗棠 周虹 程环 王瑛 QIN Dong;LI Wei-dan;SHENG Ming;GAO Dao-yuan;CHU Zong-tang;ZHOU Hong;CHENG Huan;WANG Ying(Institute of Health Service and Transfusion Medicine,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China;Facilities and Support Center,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China)

机构地区：[1]军事科学院军事医学研究院卫生勤务与血液研究所,北京100850 [2]军事科学院军事医学研究院科研保障中心,北京100850

出　　处：《军事医学》2023年第1期33-40,共8页Military Medical Sciences

基　　金：军事科学院军事医学研究院装备技术基础计划项目(202JK22003)。

摘　　要：目的考察纯化过程中不同实验条件对牛血红蛋白携氧/释氧能力的影响,筛选获得稳定携氧/释氧能力血红蛋白的方法,为血红蛋白氧载体(HBOC)的原料制备提供工艺方案。方法采用低渗溶血、微滤、阴离子层析、超滤等步骤,从新鲜牛血中分离得到纯化的牛血红蛋白。在纯化过程中,运用红细胞携氧/释氧功能评价装置对牛血红蛋白进行分析,同时检测纯化样品的高铁血红蛋白(MetHb)含量、纯度、回收率等,对纯化工艺条件进行筛选。结果研究确定较优的纯化条件为:溶血选用PB溶血液,微滤选用聚偏二氟乙烯(PVDF)材质滤膜,层析条件筛选为缓冲液A(20 mmol/L Tris-HCl,pH 8.0),缓冲液B(20 mmol/L Tris-HCl+0.15 mol/L NaCl,pH 8.0),超滤选用PBS缓冲液。该方法对牛血红蛋白的携氧/释氧能力基本无影响,分离纯化的牛血红蛋白氧饱和度达50%时的氧分压(P50)为(28.69±0.11)mmHg,变异系数(CV)为0.40%,Hill系数为2.49±0.04,CV 1.52%,纯度达到99%以上,MetHb含量低于1%。结论在纯化过程中不同实验条件可对牛血红蛋白携氧/释氧能力造成差异影响,该研究为筛选最佳工艺参数奠定了基础。Objective To study the effects of different experimental conditions on the oxygen-carrying/oxygen-releasing capacity of bovine hemoglobin in the purification process,and to screen a method for obtaining stable hemoglobin with oxygen-carrying/oxygen-releasing capacity in order to recommend a process for the preparation of raw materials for hemoglobinbased oxygen carriers(HBOCs).Methods The low-osmotic hemolysis method,microfiltration,anion exchange chromatography and ultrafiltration were used to isolate and purify bovine hemoglobin from fresh bovine blood.In the process of purification,bovine hemoglobin was analyzed with the red blood cell oxygen carrying/releasing function evaluation device,and the concentration,purity and recovery of methemoglobin(MetHb)in the purified samples were detected so as to screen the purification conditions.Results The optimal purification conditions were determined as follows:PB solution was used for hemolysis,PVDF membrane was used for microfiltration,chromatography conditions were Buffer A(20 mmol/L Tris-HCl,pH 8.0)and Buffer B(20 mmol/L Tris-HCl+0.15 mol/L NaCl,pH 8.0),and PBS buffer was used for ultrafiltration.This method had little effect on the oxygen carrying/releasing capacity of bovine hemoglobin.The P50(CV)of purified bovine hemoglobin was(28.69±0.11)mmHg(0.40%),Hill coefficient was(2.49±0.04)(1.52%),the hemoglobin protein purity exceeded 99%,and the MetHb concentration was lower than 1%.Conclusion Different experimental conditions in the purification process may have different effects on the oxygen carrying/releasing capacity of bovine hemoglobin.This study can contribute to screening the optimal process parameters.

关 键 词：携氧/释氧能力 牛血红蛋白 蛋白纯化 血红蛋白氧载体 血红蛋白测定 高铁血红蛋白 

分 类 号：R331.14[医药卫生—人体生理学]