Biver Tarita, Friani Rossella, Gattai Chiara, Secco Fernando, Tiné Maria Rosaria, Venturini Marcella
Dipartimento di Chimica e Chimica Industriale, University of Pisa, Via Risorgimento 35, 56100 Pisa, Italy.
J Phys Chem B. 2008 Sep 25;112(38):12168-73. doi: 10.1021/jp8045033. Epub 2008 Aug 30.
The equilibria and kinetics for the process of In(3+) exchange between nitrilotriacetic acid (NTA) and bovine serum transferrin (T) have been investigated in aqueous solution containing sodium bicarbonate. The metal exchange equilibria have been measured by difference ultraviolet spectroscopy at 25 degrees C, pH=7.4, and I=0.2 M (NaClO4). The acid dissociation constants of NTA and the binding constants of In(III) to NTA have also been measured. Kinetic experiments revealed that the process of In(3+) uptake by transferrin from In(NTA)2 is biphasic, the fast phase being completed in a few seconds, the slow phase lasting for hours. The fast phase has been investigated by the stopped-flow method and results in monoexponential kinetics. It involves rapid interaction of the 1:1 complex ML (M=In, L=NTA) with TB (T=transferrin, B=CO3(2-)) to give a quaternary intermediate MLTB which then evolves to an "open" MTB* ternary complex complex with expulsion of L. In turn, this complex interconverts to a "closed", more stable, form MTB. Neither the prevailing complex M2L nor the TB2 form of transferrin are directly involved in the exchange process but act as metal and protein reservoirs. The pH dependence of the reaction has been also investigated. The slow phase has not been investigated in detail; it takes several hours to go to the completeness, its slowness being ascribed to metal redistribution between the C-site and N-site of the protein, and/or metal release from polynuclear In(III) species.
在含有碳酸氢钠的水溶液中，研究了次氮基三乙酸（NTA）与牛血清转铁蛋白（T）之间In(3+)交换过程的平衡和动力学。在25℃、pH = 7.4和I = 0.2 M（高氯酸钠）条件下，通过差示紫外光谱法测定了金属交换平衡。还测定了NTA的酸解离常数以及In(III)与NTA的结合常数。动力学实验表明，转铁蛋白从In(NTA)2摄取In(3+)的过程是双相的，快速阶段在几秒钟内完成，缓慢阶段持续数小时。通过停流法研究了快速阶段，结果呈现单指数动力学。它涉及1:1络合物ML（M = In，L = NTA）与TB（T = 转铁蛋白，B = CO3(2-)）的快速相互作用，生成四元中间体MLTB，然后演变成“开放”的MTB*三元络合物并排出L。反过来，这种络合物会相互转化为“封闭”的、更稳定的MTB形式。占主导地位的络合物M2L和转铁蛋白的TB2形式都不直接参与交换过程，而是充当金属和蛋白质储存库。还研究了反应的pH依赖性。尚未详细研究缓慢阶段；它需要几个小时才能完全完成，其缓慢归因于蛋白质的C位点和N位点之间的金属重新分布，和/或多核In(III)物种释放的金属。
