人胎盘膜铁蛋白同类型双亚基功能、铁核结构和释放铁动力学研究

Functions of Double Subunits of a Type,Structure of Iron Corn, and Kinetics of Iron Release from Membrane Ferritin of Human Placenta

以人胎盘组织为实验材料,小批量制备电泳纯人胎盘膜铁蛋白(HPMF),对其结构与功能进行研究。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)技术揭示,HPMF蛋白壳由分子量分别为15 kDa(MF15)和20 kDa(MF20)的亚基组成,其中MF15蛋白含量约为MF20的3倍。经肽质量指纹图谱(PMF)技术鉴定,发现PHMF的MF15和MF20亚基与人铁蛋白(HF)L亚基均具有较高的同源性,提出HPMF由单类型但分子量不同的双亚基组成的新观点。在选用基质辅助电离激光解吸飞行时间质谱(MALDI-TOF MS)技术直接分析HPMF亚基稳定性过程中,获得5个质荷比(m/z)值为5071.25,9962.51,15131.55(MF15),19936.40(MF20)和20147.61的特征质谱峰,其对应的亚基分子式分别为[MF15]3+,[MF20]2+,[MF15]+,[MF20]+和[MF20+Fe]+。ICP-MS分析发现,HPMF铁核中的铁和无机磷酸盐(Pi)含量仅为85Fe3+/HPMF和15Pi/HPMF,其中Fe3+/Pi比值约为5.72,明显低于绝大多数哺乳动物铁蛋白,但却高于细菌铁蛋白(BF)。在释放铁动力学方面,HPMF以一级反应动力学途径释放完整铁核中的铁量,其释放时间约需750 min,其释放铁的速率慢于马脾铁蛋白(HSF)和猪胰铁蛋白(PPF)(约60 min)。根据HPMF完全不同于大部分哺乳动物、植物和细菌铁蛋白的新颖结构与功能,提出HPMF在母体和胎儿之间中起着释放、储存和转运铁的复合生理功能的铁转运模型。

As an experimental material of human placental tissue,membrane ferritin of human placenta（HPMF） with electrophoresis purity was prepared in batch.SDS-PAGE approach reveals that the protein shell consists of double subunits of a type in HPMF,which the molecular weights of both subunits were calculated to be approximately 15 and 20 kDa respectively,named MF15 and MF20.In addition,the protein content of MF15 is about three times more than that of M20 in SDS-PAGE gel.Using the approach of peptide mass fingerprinting（PMF）,both MF15 and MF20 subunits were identified to show the high homology with reference to L subunit of human ferritin,pointing out that HPMF was a novel ferritin of two subunits involving in single type and different molecular weight.Using a matrix-assisted laser desorption ionization time of flight mass spectrometry to investigate the subunit-stability of HPMF,five mass peaks corresponding ratio（m/z） of mass to charge such as 5071.25,9962.51,15131.55（MF15）,19936.40（MF20） and 20147.61 were indicated to have five subunit formula of [MF15]^3＋,[MF20]^2＋,[MF15]^＋,[MF20]^＋and[MF20＋Fe]^＋,respectively.Using inductively coupled plasma-mass spectrometry to analyze the elemental composition of HPMF,only 85 Fe^3＋ and 15 inorganic phosphate（Pi） in the iron core of per molecular of HPMF were found,which was significantly lower than that of most mammal ferritins,but higher than that of bacterial ferritin（BF）.Kinetic studies showed that iron release from the complete HPMF followed the law with first-order reaction,which also differed from that with two different rates in mammal ferritins.In addition,the time of the iron release completely needed about 750 min in human placental membrane ferritin（HPMF）,which was longer than 60 min in both horse spleen ferritin（HSF） and pig pancreatic ferritin（PPF）.Accordingly,based on the difference of structure and function among HPMF,mammalian ferritins,plant ferritins,and BF,we proposed M15 and M20 subunit play different role in iron transporting from maternal blood to fetal blood.