CALORIMETRIC STUDIES OF THE BINDING OF FERRIC IONS TO OVOTRANSFERRIN AND INTERACTIONS BETWEEN BINDING-SITES
CALORIMETRIC STUDIES OF THE BINDING OF FERRIC IONS TO OVOTRANSFERRIN AND INTERACTIONS BETWEEN BINDING-SITES
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DOI：
10.1021/bi00114a008
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发表时间：
1991-12-17
期刊：
影响因子：
2.9
通讯作者：
BRANDTS, JF
中科院分区：
文献类型：
--
作者：
LIN, LN;MASON, AB;BRANDTS, JF
转铁蛋白是两个结构域的蛋白质，每个结构域都有一个很强的铁结合部位。用超灵敏滴定量热法，详细研究了铁离子(与2倍摩尔过量的氮三乙酸酯络合)与转铁蛋白的两个位置的结合以及与N端和C端半分子中单一位置的结合。在过量的碳酸氢根离子存在下，结合分两个动力学步骤进行。接触结合的快速过程相对于仪器响应时间是瞬时的，对于N位是强放热的，而对于C位是较小的放热过程，对应于螯合铁离子的结合。碳酸氢盐插入伴随着氮三乙酸酯的释放的较慢过程在7-37℃的温度范围内发生在2-20分钟的时间尺度上，并且对于N位是吸热的，对于C位是放热的，在C位的插入速度明显较慢。对于这两个位置，结合的Delta-H强烈依赖于温度，这是由大的负结合Delta-C(P)引起的，这可能表明疏水基团从与水的接触中移除。当碳酸氢根离子不存在时，只能看到接触结合的快速过程。尽管检测到数量上的差异，但半分子内的每个位置在性质上与完整的卵转铁蛋白中的相同位置相似。结果表明，随着Fe+3在N和C位之间的自由交换，与卵转铁蛋白的接触结合是可逆的，而插入小苏打后，与任一位点的结合基本上是不可逆的。与C位相比，第一个铁离子与N位结合的强烈偏好是由于其较大的接触结合常数和较快的碳酸氢盐插入速度，而不是由于碳酸氢盐插入后更强的热力学结合。真正的平衡只有在更长的时间内才能实现。在另一系列实验中，在碳酸氢钠存在下，两个半分子在不同的连接状态下与Fe+3进行了直接结合研究。结果表明，卵转铁蛋白中的两个结合位点之间的距离约为40埃，它们并不是相互独立的，而是由于结构域-结构域相互作用的热和自由能随配体的变化而相互联系的结果。这些相互作用的变化是这样的，相对于没有结构域间信号的情况，它们增加了铁离子与N位的热力学结合常数，降低了与C位的结合常数。这可能有利于也可能不利于卵转铁蛋白的生理功能。
Transferrins are two-domain proteins with a very strong site for iron binding located in each domain. Using ultrasensitive titration calorimetry, the binding of ferric ion (chelated with a 2-fold molar excess of nitrilotriacetate) to the two sites of ovotransferrin was studied in detail as well as the binding to the single site in the N- and C-terminal half-molecules. In the presence of excess bicarbonate ion, the binding occurs in two kinetic steps. The fast process of contact binding is instantaneous with respect to instrument response time, is strongly exothermic for the N site and less so for the C site, and corresponds to binding of the chelated ferric ion. The slower process of bicarbonate insertion with concomitant release of nitrilotriacetate occurs on a time scale of 2-20 min over the temperature range 7-37-degrees-C and is endothermic for the N site and exothermic for the C site, with rates being significantly slower for insertion at the C site. The DELTA-H of binding is strongly temperature-dependent for both sites, arising from a large negative DELTA-C(p) of binding which probably indicates removal of hydrophobic groups from contact with water. When bicarbonate ion is absent, only the fast process of contact binding is seen. Each site within a half-molecule is qualitatively similar to the same site in intact ovotransferrin, although quantitative differences were detected. It was shown that contact binding to ovotransferrin occurs reversibly with free exchange of Fe+3 between N and C sites, while the attachment to either site becomes essentially irreversible after bicarbonate insertion. The strong preference for the first ferric ion to bind to the N site is shown to be due to its larger contact binding constant and the faster rate of bicarbonate insertion, relative to the C site, and is not due to stronger thermodynamic binding after bicarbonate insertion. True equilibrium is achieved only over much longer periods of time. In another series of experiments, direct binding studies were carried out between the two half-molecules under different states of ligation with Fe+3 in the presence of bicarbonate. The results indicate that the two binding sites in ovotransferrin, separated by ca. 40 angstrom, are not independent of one another but communicate as a result of ligand-dependent changes in the heats and free energies of domain-domain interactions. These changes in interactions are such that they increase the thermodynamic binding constant of ferric ion to the N site and decrease the binding constant to the C site, relative to the situation which would exist in the absence of interdomain signaling. This may or may not be advantageous in carrying out the physiological function of ovotransferrin.