Bezwoda W R, Mansoor N
Department of Medicine, Haematology/Oncology, University of the Witwatersand Medical School, Johannesburg, South Africa.
Biomed Chromatogr. 1989 May;3(3):121-6. doi: 10.1002/bmc.1130030307.
The isolation and properties of lactoferrin from human breast milk and from neutrophilic granulocytes were investigated. Human breast milk lactoferrin was purified by means of heparin-sepharose or Cibacron Blue affinity chromatography. Quantitative recovery using these two methods was comparable but Cibacron Blue affinity chromatography allowed for isolation of a more homogenous protein. Lactoferrin could only be isolated from human neutrophilic granulocytes by sequential use of antibody affinity followed by non-specific affinity chromatography. Both breast milk lactoferrin and granulocyte lactoferrin were separated into apo and iron-rich species by SDS polyacrylamide gel chromatography. Iron binding is accompanied by a conformational change in tertiary structure associated with more rapid electrophoretic migration. The isoelectric point of both human breast milk lactoferrin and human granulocyte lactoferrin is 5.5-6.2. Both types of lactoferrin have similar iron binding properties with release of iron from the one binding site occurring at pH 5.2-6.0 while the other binding site holds on to iron down to pH 3.6-3.2. Despite the high affinity for iron the percentage saturation of native lactoferrin is low, that for breast milk lactoferrin averaging 12-25% and that for granulocyte lactoferrin less than 10%.
对从人母乳和嗜中性粒细胞中分离乳铁蛋白及其性质进行了研究。人母乳乳铁蛋白通过肝素 - 琼脂糖或汽巴克隆蓝亲和色谱法进行纯化。使用这两种方法的定量回收率相当，但汽巴克隆蓝亲和色谱法能分离出更均一的蛋白质。乳铁蛋白只能通过依次使用抗体亲和色谱法和非特异性亲和色谱法从人嗜中性粒细胞中分离出来。通过SDS聚丙烯酰胺凝胶色谱法，母乳乳铁蛋白和粒细胞乳铁蛋白都被分离成脱铁和富铁形式。铁结合伴随着三级结构的构象变化，这与更快的电泳迁移相关。人母乳乳铁蛋白和人粒细胞乳铁蛋白的等电点均为5.5 - 6.2。两种类型的乳铁蛋白具有相似的铁结合特性，在pH 5.2 - 6.0时，一个结合位点的铁会释放，而另一个结合位点在pH 3.6 - 3.2时仍能保留铁。尽管对铁具有高亲和力，但天然乳铁蛋白的饱和百分比很低，母乳乳铁蛋白平均为12 - 25%，粒细胞乳铁蛋白则小于10%。
