基于FⅧ探讨不同脱盐方式的效果及对其成分的影响

Desalination effect on FⅧcomponents:a compartive study among 5 desalination methods

目的基于人凝血因子Ⅷ(human coagulation factorⅧ,FⅧ)比较5种脱盐方法的脱盐效果及对FⅧ成分的影响,为蛋白脱盐方式提供参考。方法分别用Sephadex G-25 Medium凝胶、Fractogel EMD BioSEC凝胶、超滤、室温透析和4℃透析5种方法对人FⅧ进行脱盐处理。通过Na^(+)、柠檬酸根离子、甘氨酸去除率评估脱盐效果。通过脱盐前后FⅧ蛋白回收率、FⅧ活性(coagulation factorⅧactivity,FⅧ∶C)、VWF抗原(VWF antigen,VWF∶Ag)、VWF活性(VWF activity,VWF∶Ac)、VWF多聚体及SDS-PAGE分析,评估其对FⅧ成分的影响。结果在脱盐效果方面:Na+在超滤脱盐时去除率最低,为(97.90±0.06)%,Fractogel EMD BioSEC凝胶脱盐去除率最高,为(99.82±0.07)%。除Sephadex G-25 Medium凝胶脱盐与Fractoge EMD BioSEC凝胶脱盐间Na^(+)去除率无统计学意义(P=0.90)外,其他4种方法间Na+去除率存在统计学意义。甘氨酸在超滤脱盐时去除率最低,为(95.78±0.42)%,Fractogel EMD BioSEC凝胶脱盐去除率最高,为(99.81±0.08)%。除超滤脱盐外,其他4种脱盐方法间甘氨酸去除率无统计学意义。柠檬酸根离子在5种方法间去除率均无统计学意义(P=0.85)。对于FⅧ成分的影响方面:超滤脱盐FⅧ∶C、VWF∶Ag、VWF∶Ac及蛋白回收率最高,分别为(18.34±1.99)IU/mL、(11.81±0.33)IU/mL、(12.26±0.58)IU/mL、(97.13±1.37)%。5种方法脱盐前后VWF∶Ac/VWF∶Ag无明显变化。SDS-PAGE及VWF多聚体分析表明,不同脱盐方法对蛋白组成种类影响不明显。结论尽管不同方式脱盐对FⅧ蛋白组成种类无明显影响,但脱盐效果存在差异,不同方式脱盐对蛋白回收率、FⅧ∶C、VWF∶Ag及VWF∶Ac影响显著。蛋白处理过程中,对于脱盐方式的选择应给予足够重视。

Objective To compare the desalination effects of five desalination methods and their effects on the components for human coagulation factorⅧ(FⅧ),and provide reference for selection of protein desalination methods.Methods Sephadex G-25 Medium gel,Fractogel EMD BioSEC gel,ultrafiltration,room temperature dialysis and 4℃dialysis were used to desalt human FⅧ.The desalination effect was evaluated by the removal rate of Na+,citrate ion and glycine.FⅧprotein recovery,FⅧactivity(FⅧ∶C),VWF antigen(FⅧVWF∶Ag),VWF activity(VWF∶Ac),VWF polymers and SDS-PAGE analysis before and after desalination were compared to evaluate the effect of desalination on FⅧcomponents.Results In terms of desalination effect,the removal rate of Na+was the lowest in ultrafiltration desalination,while that of Fractogel EMD BioSEC gel was the highest[(97.90±0.06)%vs(99.82±0.07)%].Except that there was no statistical significance between Sephadex G-25 Medium gel desalination and Fractogel EMD BioSEC gel desalination(P=0.90),the removal rates of the other four methods were statistically significant.The removal rate of glycine was the lowest in ultrafiltration desalination,wihle that of Fractogel EMD BioSEC gel desalination was the highest[(95.78±0.42)%vs(99.81±0.08)%].Significant difference in glycine removal was noticed in ultrafiltration desalination,but not among the other four desalination methods.There was no significant difference in the removal rate of citrate ions among the five methods(P=0.85).For the effect of FⅧcomponents,FⅧ∶C,VWF∶Ag,VWF∶Ac and protein recovery rates of ultrafiltration desalination were the highest,with(18.34±1.99)IU/mL,(11.81±0.33)IU/mL,(12.26±0.58)IU/mL and(97.13±1.37)%,respectively.There was no significant change in VWF∶Ac/VWF∶Ag before and after desalination by the five methods.SDS-PAGE and VWF polymer analysis showed that different desalination methods had no significant impact on protein composition.Conclusion Although different desalination methods had no significant effect on the composition of FⅧprotein,the desalination effect was different.Moreover,different desalination methods had significant effects on protein recovery,FⅧ∶C,VWF∶Ag and VWF∶Ac.The selection of desalination methods should be more considered during protein processing,