螯合亲和色谱法分离转铁蛋白的4种分子形式

High-Performance Immobilized Metal Ion Affinity Chromatographic Separation of Four Types of Molecules of Transferrin

在ＴＳＫｇｅｌ－Ｃｈｅｌａｔｅ－５ＰＷ柱上用固定Ｃｕ２^（２＋）离子的螯合亲和色谱法（ＨＰ－ＩＭＡＣ）研究了Ｆｅ（Ⅲ）同去铁转铁蛋白的结合，讨论了所固定的几种金属离子对保留时间和柱寿命的影响；在生理ｐＨ条件下分离出了非饱和铁转铁蛋白的４种分子形式：ＦｅＮＴｆ，ＴｆＦｅｃ，ＦｅＮＴｆＦｅｃ（或Ｆｅ２Ｔｆ）和Ａｐｏ－Ｔｆ。

he binding of Fe(Ⅱ) by Apo-transferrin has been investigated with high-performance immobilized metal ion affinity chromatography on a Cu (Ⅱ)-loaded TSK gel Chelate-5PW column by elution with linear gradient of imidazole under phymological PH condition.The influences of several loaded metals on the retention time and the column life were discussed. The column life and the affinity of the protein for the immobilized metal ions follow the order Cu￣(2+) >Ni￣(2+)>Zn￣(2+) and the Cu(Ⅱ)-loaded column gives the best resolution. This elution procedure made it possible to use the column over 100 runs when the metal ion Cu￣(2+) is still immobilized.Under physiological PH condition the transferrin (Tf) which contains iron (not iron-saturated) has been separated into four types of molecules: Fe_NTf, the binding of Fe(Ⅲ) to Tf at the N-terminal. domain I Tffe_c, the binding of Fe(Ⅲ) to Tf at the C-terminal domain f Fe_N TfFe_c (or Fe_2Tf), the binding of Fe(Ⅲ) to Tf on both sites;and Apo-Tf.The foundation of separation method of the four forms of molecules of human transferrin under the physiological PH condition has a great importance to the studies of the binding of the trace ferric iron and other trace metals to human transferrin and their physiological effect.