高铁含量猪血红蛋白的提取纯化及特性研究
Purification of iron-rich swine hemoglobin and its characteristics
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摘要: 取新鲜猪血红细胞裂解原液依次用10%、20%、30%、40%、50%、60%饱和度的硫酸铵沉淀,并分步收集沉淀,获得粗猪血红蛋白。结果表明,血红蛋白主要集中于50%和60%饱和度的硫酸铵沉淀中,杂蛋白主要集中在10%40%饱和度的硫酸铵沉淀。50%和60%饱和度的硫酸铵沉淀后获得的粗猪血红蛋白用DEAE-Sephrose FF阴离子交换色谱进一步分离,均获得了单一蛋白峰,对出峰处各管样品进行SDS-PAGE电泳,均为分子量为16.4 ku的单一条带,为猪血红蛋白的单亚基分子量,经PDQuest软件分析纯度为95.2%,比市售的猪血红蛋白纯度（约为85%）高。采用Bradford法测定各步分离组分的蛋白浓度,结果表明,血红蛋白得率占总蛋白的49.69%;纯化猪血红蛋白的溶解度为32.96%;纯化后猪血红蛋白的含铁量为0.335%,与血红蛋白中理论铁含量0.35%接近。Abstract: Swine hemoglobin was extracted from fresh lysed swine red blood cell. The lysed solution was precipitated by 10%,20%,30%,40%,50%,and 60% saturated ammonium sulfate,and precipitaton from each step was collected and the cruel swine hemoglobin was obtained. The results showed the impure protein was mainly distributed in 10%~40% saturated ammonium sulfate pelleted precipitations, while the swine hemoglobin was mainly distributed in the precipitation obtained from 50% and 60% saturated ammonium sulfate precipitated production and was separated by the DEAE-Sepharose FF anion material and SDS-PAGE was used to identify their purification. The results of DEAE-Sepharose FF showed that both of the separated dilution got single protein peak. The SDS-PAGE results showed that the hemoglobin was mainly concentrated by 50% and 60% saturated ammonium sulfate.The molecular weight of the purified hemoglobin was 16.4 ku which was in accordance with with that of the monomer of hemoglobin. The purity of the purified hemoglobin was 95.2% canalyzed by PDQuest,which was higer than that of the purchased porcine hemoglobin whose purity was 85%. The protein concentration from each step was determined by Bradford method. The result showed that obtained rate of hemoglobin was accounted for 49.69% in the total protein fractions. The solubility of purified porcine hemoglobin was 32.96%. The iron content in purifiend hemoglobin was 0.335% whicn was closed to the theory content of natural hemoglobin.
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