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脱离了红细胞的血红蛋白(Hb)在溶液中易被氧化成高铁血红蛋白(MetHb),失去载氧活性.实验发现,当对红细胞裂解液进行微孔膜分离时,高铁血红蛋白增加不多;但在用层析法去除超氧化物歧化酶等其他红细胞蛋白后再进行超滤膜浓缩血红蛋白时,则出现较多的血红蛋白被氧化成高铁血红蛋白的现象,其氧化反应随超滤过程膜表面流体切向速度的增加而加快,随溶液温度的增加而加快.抗氧化剂的存在能有效地降低高铁血红蛋白的生成,谷胱甘肽(GSH)、半胱氨酸、N-乙酰半胱氨酸、亚硫酸钠、抗坏血酸(Vc)在溶液中和超滤过程中都能起到对血红蛋白载氧活性的保护作用.其中Vc的效果最好,最适加入量(摩尔比)Vc/Hb=8,pH8.将抗氧化的优化条件整合到从红细胞裂解液开始到超滤浓缩血红蛋白的整个流程,在离子交换层析后,添加Vc作为抗氧化剂进行超滤浓缩,Hb活性得到了很好的保护,MetHb的含量控制在2.3%,成功地制备了低MetHb含量的纯化血红蛋白.
Hemoglobin (Hb), which is separated from erythrocytes, is easily oxidized to methemoglobin (MetHb) in solution and loses the activity of oxygen-loading.It has been found that there is not much increase of methemoglobin when the red blood cell lysate is microporous membrane separation, Chromatography to remove superoxide dismutase and other red blood cell proteins and then ultrafiltration membrane hemoglobin concentration, then there are more hemoglobin is oxidized to methemoglobin, the oxidation reaction with the ultrafiltration membrane surface fluid tangential velocity And accelerated with the increase of solution temperature.The presence of antioxidants can effectively reduce the formation of methemoglobin, glutathione (GSH), cysteine, N-acetylcysteine, sodium sulfite, ascorbic acid (Vc) can protect hemoglobin oxygen-carrying activity both in solution and in ultrafiltration process.Vc has the best effect, the optimum dosage (molar ratio) Vc / Hb = 8, pH 8. Antioxidant Of the optimal conditions integrated into the whole process from the red blood cell lysate to ultrafiltration concentrated hemoglobin, after ion exchange chromatography, adding Vc as anti-oxidant enrichment by ultrafiltration, Hb activity was very good Protection, MetHb control the content of 2.3%, purified successfully MetHb low hemoglobin contents were prepared.