Cheng Y, Mason A B, Woodworth R C
Department of Biochemistry, University of Vermont, Burlington 05405, USA.
Biochemistry. 1995 Nov 14;34(45):14879-84. doi: 10.1021/bi00045a032.
The binding of the two synergistic anion mimics, phosphate and sulfate, and of the synergistic anions, malonate and oxalate, to the N-lobe of recombinant human serum transferrin (hTF/2N) wild-type and H207E mutant protein was assessed by difference ultraviolet (UV) spectroscopy at 246 nm as a function of pH. The absolute values of both the maximum delta epsilon 246 and the Kd decreased with decreasing pH. A plot of -log Kd vs pH gave a straight line with a slope of -1.0. Furthermore, the sum of -log Kd and pH is a constant for each anion binding to each protein. We interpret these data to mean that each anion binds in divalent form along with an H+. The binding equilibrium then appears to be H+ + hTF/2N + X2- reversible K' H-hTF/2N(X) and log K' = -log Kd + pH. A plot of delta epsilon 246 vs pH was sigmoidal with a pKa = 7.4 for both proteins with phosphate and sulfate. When synergistic anions were used with hTF/2N, malonate and oxalate gave pKas of ca. 6.9 and 7.1 for dependence of delta epsilon 246 on pH, but values of 7.3 and 7.6 for the H207E mutant protein. In an attempt to locate the anion binding site in hTF/2N, the binding of sulfate to the single point mutants of the N-lobe of human transferrin, K296E, K296Q, and K206Q, was carried out by difference UV spectroscopy at pH 7.4. In the case of K296E, sulfate binding gave delta epsilon 246 = 0, while for K296Q, it gave a slightly positive delta epsilon 246.(ABSTRACT TRUNCATED AT 250 WORDS)
通过在246 nm处的差示紫外光谱法，测定了两种协同阴离子模拟物（磷酸盐和硫酸盐）以及协同阴离子（丙二酸盐和草酸盐）与重组人血清转铁蛋白（hTF/2N）野生型和H207E突变体蛋白N叶的结合情况，该结合情况是pH的函数。最大Δε246和Kd的绝对值均随pH降低而减小。-log Kd对pH作图得到一条斜率为-1.0的直线。此外，对于每种阴离子与每种蛋白质的结合，-log Kd与pH之和为常数。我们将这些数据解释为每种阴离子以二价形式与一个H⁺结合。结合平衡似乎是H⁺ + hTF/2N + X²⁻ ⇌ K' H-hTF/2N(X)，且log K' = -log Kd + pH。对于磷酸盐和硫酸盐，Δε246对pH作图呈S形，两种蛋白质的pKa均为7.4。当协同阴离子与hTF/2N一起使用时，丙二酸盐和草酸盐的Δε246对pH的依赖性给出的pKa约为6.9和7.1，但H207E突变体蛋白的值为7.3和7.6。为了确定hTF/2N中的阴离子结合位点，通过在pH 7.4下的差示紫外光谱法，测定了硫酸盐与人转铁蛋白N叶单点突变体K296E、K296Q和K206Q的结合情况。在K296E的情况下，硫酸盐结合给出的Δε246 = 0，而对于K296Q，它给出的Δε246略为正值。（摘要截短至250字）
