鲨鱼肝铁蛋白亚基解离与组装机理的研究

Dissociation and Recombination Mechanism of Ferritin Subunits in the Liver of Sphyrna Zygaena

小批量制备质谱纯鲨鱼肝铁蛋白(Liver Ferritin of Sphyrna zygaena,SZLF)。在弱酸介质(pH1.0)中,天然电泳结果显示,SZLF蛋白质亚基20 min后开始解离。选用透射电子显微镜跟踪SZLF亚基解离与重组装全过程和蛋白壳与铁核尺寸变化,发现SZLF在亚基酸解离过程中,随着pH值的降低,铁核和蛋白壳的尺寸呈现相同的变化趋势,这种变化趋势可能与铁核内层铁的释放和蛋白壳的解离与去折叠有关。SZLF蛋白壳的重组装过程则是一个快速过程,并且是由松散熔球态向紧密态转变的过程。SZLF由单类型亚基组成,而马脾铁蛋白(Horse Spleen Ferritin,HSF)由H和L两种亚基类型组成。在基质pH3.0条件和激光辅助下,混合HSF和SZLF仍然可释放各自的亚基且形成准亚基离子,供基质辅助激光解析电离飞行时间质谱分析,说明此时SZLF的亚基间相互作用强度减弱但并没有去折叠。TEM技术在铁蛋白解离和重组装过程中的应用,为进一步研究铁蛋白纳米包装的过程和机理提供新颖的、可行的和更加直观的研究手段。

Liver ferritin of Sphyrna zygaena（SZLF） with purity of mass spectrum was prepared in batch.Under the condition of acidifying medium at pH 1.0,PAGE showed that SZLF subunits treated for 20 min began to dissociate.A whole process of subunit dissociation and recombination was monitored by transmission electron microscopy（TEM）.In addition,the changes of size of both protein shell and iron core were also determined by TEM directly.It was found that in the acid dissociation process of SZLF subunits,the size of iron core and protein shell showed the same trend of change,which might be related to not only the iron release of inner iron core but the dissociation and unfolding of the protein shell.The passway of SZLF recombination is a fast step,which is a conversion process from incompact moltenglobule to compact ferritin.Under the assistant of matrix acidity pH 3.0 and laser,SZLF mixed with horse spleen ferritin（HSF） still has capacity to release its subunits to form subunit ions for mass analysis by a MALDI-TOF mass spectrometer,which indicates that the interaction intensity between the subunits was weaken but they were not unfolded under this pH condition.TEM technology can be applied in studying both dissociation and recombination in ferritin subunits.