Harris W R, Stenback J Z
Department of Chemistry, University of Idaho, Moscow 83843.
J Inorg Biochem. 1988 Jul;33(3):211-23. doi: 10.1016/0162-0134(88)80050-3.
The binding of zinc(II) to human serum transferrin has been studied as a function of the solution concentration of sodium bicarbonate in 100 mM, pH 7.4 hepes buffer at 25 degrees C. The apparent molar absorptivity of the zinc-transferrin complex has been determined from the initial slopes of titration curves of delta epsilon versus the ratio of [Zn]/[Tf]. This absorptivity represents the difference between the positive absorbance of the ternary Zn-HCO3-Tf species in the sample cuvette and the negative absorbance of binary HCO3-Tf species in the reference cuvette. Higher concentrations of bicarbonate increase the degree of saturation of apo-Tf with bicarbonate and thus increase the apparent absorptivity of the zinc-Tf complex. Titrations of apo- and monoferric transferrins with bicarbonate indicate that there is little, if any, difference in the bicarbonate binding constants of the two specific transferrin binding sites. An equilibrium constant of log K = 2.49 has been used to calculate the degree of saturation of the C-terminal binding site with bicarbonate. The zinc-binding affinity of this site depends linearly on this degree of saturation. The scatter in the zinc-binding constants of the weaker N-terminal site precludes a similar analysis of the bicarbonate-dependence of binding at this site. The results strongly support the previous proposal that binding of the synergistic bicarbonate anion is responsible for the uv absorption observed upon addition of bicarbonate to apoTf.
在25℃下，于100 mM、pH 7.4的羟乙基哌嗪乙磺酸（HEPES）缓冲液中，研究了锌（II）与人血清转铁蛋白的结合情况，该结合情况是碳酸氢钠溶液浓度的函数。锌 - 转铁蛋白复合物的表观摩尔吸光系数是根据δε与[Zn]/[Tf]比值的滴定曲线的初始斜率确定的。该吸光系数代表样品比色皿中三元Zn - HCO₃ - Tf物种的正吸光度与参比色皿中二元HCO₃ - Tf物种的负吸光度之间的差异。较高浓度的碳酸氢盐会增加脱铁转铁蛋白（apo - Tf）与碳酸氢盐的饱和程度，从而增加锌 - Tf复合物的表观吸光系数。用碳酸氢盐滴定脱铁转铁蛋白和单铁转铁蛋白表明，两个特定转铁蛋白结合位点的碳酸氢盐结合常数几乎没有差异（如果有差异也很小）。log K = 2.49的平衡常数已用于计算C端结合位点与碳酸氢盐的饱和程度。该位点的锌结合亲和力与此饱和程度呈线性关系。较弱的N端位点的锌结合常数存在分散性，这使得无法对该位点结合的碳酸氢盐依赖性进行类似分析。结果有力地支持了先前的提议，即协同碳酸氢根阴离子的结合是向脱铁转铁蛋白中添加碳酸氢盐时观察到的紫外吸收的原因。
