人源性PolyPHb-SOD-CAT的制备及其抗氧化特性的初步研究

The preparation of anthropogenic PolyPHb-SOD-CAT and the preliminary study on its oxidization resistance

目的通过交联血红蛋白、超氧化物歧化酶和过氧化氢酶,制备具有抗氧化特性的人源性聚合血红蛋白,表征其基本理化性质,并对其抗氧化特性进行初步研究。方法使用戊二醛（GDA）交联血红蛋白（Hb）和抗氧化酶,分别通过圆二色谱和氧解离曲线分析蛋白质二级结构及血红蛋白携氧功能的变化;采用试剂盒检测超氧化物歧化酶（SOD）和过氧化氢酶（CAT）活性;通过体外保存实验初步评估其抗氧化性。结果制备的抗氧化酶交联的聚合人脐带血红蛋白溶液Hb浓度（20±1）g/L,高铁血红蛋白浓度（Met Hb,%）（4.98±0.61）%,SOD活性（23 601.91±2 421.14）U/gHb,CAT活性（536.06±42.43）U/gHb;圆二色谱结果显示,戊二醛交联未使蛋白二级结构发生明显改变;氧解离曲线结果显示,戊二醛交联提高了血红蛋白的氧亲和力;PolyPHb-SOD-CAT与聚合人胎盘血红蛋白（PolyPHb）相比,在25℃保存期间,前者Met Hb含量更低。结论制备出具有抗氧化酶活性的PolyPHb-SOD-CAT,其蛋白二级结构发生改变氧亲和力增加,在体外实验中表现出了抗氧化特性,可作为1种具有抗氧化特性的血红蛋白类载氧体。

Objective To prepare oxidization-resistant polyhemoglobin by cross-linking hemoglobin,superoxide dismutase and catalase. We aim to analyze its basic physicochemical properties and oxidization resistance capability. Methods Using glutaraldehyde as the crosslinker to crosslink hemoglobin with antioxidant enzymes; The secondary structure of protein and the function of hemoglobin were detected through circular dichroism（CD） spectra analysis and oxygen dissociation curve analysis respectively. The activities of superoxide dismutase（SOD） and catalase（CAT） in the PolyPHb-SOD-CAT were also detected by respective kit. The anti-oxidability of PolyPHb-SOD-CAT was assessed by in-vitro conservation experiments. Results Polymerized human placenta hemoglobin-superoxide dismutase-catalase（PolyPHb-SOD-CAT） was successfully prepared by the following formula：（1） Hemoglobin（g/d L） ： 20±1;（2） methemoglobin（%） ： 4. 98±0. 61;（3） SOD（U/gHb） ： 23 601. 91±2 421. 14;（4） CAT（U/ml） ： 536. 06±42. 43. The results of CD showed significant changes on the secondary structure of the protein; The results of oxygen-binding cures showed that the oxygen affinity of hemoglobin was raised after crosslinking by glutaraldehyde;A lower percentage of methemoglobin was produced during the 25℃ conservation experiments in comparison to PolyPHb which was not crosslinked with antioxidant enzymes. Conclusion PolyPHb-SOD-CAT with the activity of antioxidant enzymes was prepared with anti-oxidability in vitro experiments. The secondary structure of the protein was altered and the oxygen affinity of hemoglobin was raised.