Kinetic studies on the removal of iron and aluminum from recombinant and site-directed mutant N-lobe half transferrins.
Kinetic studies on the removal of iron and aluminum from recombinant and site-directed mutant N-lobe half transferrins.
复制标题
从重组和定点突变 N 叶半转铁蛋白中去除铁和铝的动力学研究。
DOI：
10.1021/bi9810454
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Brown,T
中科院分区：
文献类型：
--
作者：
Li,Y;Harris,WR;Maxwell,A;MacGillivray,RT;Brown,T
在pH 7.4 Hepes缓冲液中，25°C进行了动力学研究，研究了从重组人血清转铁蛋白（Tf/2N） n -叶半分子和该蛋白的R124A、K206A和K296A突变体中去除Fe（III）和Al（III）的过程。3-羟基吡啶-4-酮（去铁酮）和硝基三乙酸（NTA）对Tf/2N中铁的去除率与n端单铁转铁蛋白（Tf- fen）的去除率基本相同。对于Tf/2N和Tf- fen，去铁蛋白脱除铁遵循简单的饱和动力学，而NTA脱除铁遵循简单的一级动力学。两种蛋白质在PPi去除铁方面存在一些差异，但这可能是由于不同研究中氯化物浓度的差异。在碳酸氢盐的环境浓度下，将Fe(NTA)2加入R124A形成Fe - NTA - Tf三元配合物，但通常的Fe - CO3 - Tf配合物可以在碳酸氢盐过量的情况下加入铁离子形成。这种配合物通过一阶机制快速释放铁。这表明金属从转铁蛋白中释放的一级组分涉及到协同碳酸盐阴离子的位移。由于铁在pH 7.4下从K206A和K296A中去除非常缓慢，因此对更不稳定的Tf/2N、K206A和K296A的Al3+配合物进行了研究。pp3对Tf/2N中Al3+的去除遵循与铁去除中观察到的配体浓度相同的复杂动力学顺序，而对K206A和K296A中Al3+的去除则恢复到简单的饱和过程。高氯酸盐的加入阻碍了K206A和K296A中Al3+的去除，这表明这些赖氨酸残基与无机阴离子对金属去除率的变构效应无关。
Kinetic studies have been conducted in pH 7.4 Hepes buffer at 25 °C on the removal of Fe(III) and Al(III) from the recombinant N-lobe half molecule of human serum transferrin (Tf/2N) and from the R124A, K206A, and K296A mutants of this protein. The rates of iron removal from Tf/2N by 3-hydroxypyridin-4-one (deferiprone) and nitrilotriacetic acid (NTA) are essentially identical with previous results on N-terminal monoferric transferrin (Tf-FeN). For both Tf/2N and Tf-FeN, iron removal by deferiprone follows simple saturation kinetics, while iron removal by NTA follows simple first-order kinetics. There is some discrepancy between the two proteins with respect to iron removal by PPi, but this may be due to differences in the chloride concentrations among different studies. The addition of Fe(NTA)2to R124A at ambient bicarbonate concentrations forms the Fe−NTA−Tf ternary complex, but the usual Fe−CO3−Tf complex can be formed by adding ferrous ion in the presence of a larger excess of bicarbonate. This complex releases its iron very rapidly by a mechanism that is first-order with respect to the ligand. This suggests that the first-order component of metal release from transferrin involves the displacement of the synergistic carbonate anion. Since iron removal from K206A and K296A at pH 7.4 is extremely slow, studies have been conducted on the more labile Al3+complexes of Tf/2N, K206A, and K296A. The removal of Al3+from Tf/2N by PPifollows the same complex kinetic order with respect to the ligand concentration that is observed for iron removal, while the removal of Al3+from both K206A and K296A reverts to a simple saturation process. The addition of perchlorate retards the removal of Al3+from both K206A and K296A, suggesting that these lysine residues are not associated with the allosteric effects of inorganic anions on the rates of metal removal.