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首页> 外文期刊>Biochemistry >PH DEPENDENCE OF SPECIFIC DIVALENT ANION BINDING TO THE N-LOBE OF RECOMBINANT HUMAN TRANSFERRIN
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PH DEPENDENCE OF SPECIFIC DIVALENT ANION BINDING TO THE N-LOBE OF RECOMBINANT HUMAN TRANSFERRIN

机译：特定价阴离子与重组人转铁蛋白N原子结合的pH依赖性

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摘要

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 K-d decreased with decreasing pH. A plot of -log K-d vs pH gave a straight line with a slope of -1.0. Furthermore, the sum of -log K-d 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 + X(2-) 6 H-hTF/2N(X) and log K' = -log K-d + pH. A plot of Delta epsilon(246) vs pH was sigmoidal with a pK(a) = 7.4 for both proteins with phosphate and sulfate. When synergistic anions were used with hTF/2N, malonate and oxalate gave pK(a)s 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). For K206Q, the binding gave a logK' of 10.98, which is 0.6 units less than the constant obtained from sulfate binding to hTF/2N wild-type protein. These data show that these two lysine residues have an important role in divalent anion binding. [References: 27]
机译：通过差异紫外线评估了两种协同阴离子模拟物磷酸根和硫酸根以及协同阴离子丙二酸和草酸与重组人血清转铁蛋白（hTF / 2N）野生型和H207E突变蛋白的N叶的结合。 （UV）光谱在246 nm下随pH的变化而变化。随pH值的降低，最大δepsilon（246）和K-d的绝对值均降低。 -log K-d对pH的曲线给出了斜率为-1.0的直线。此外，对于每个与每种蛋白质结合的阴离子，-log K-d和pH的总和是恒定的。我们将这些数据解释为意味着每个阴离子都以二价形式与H +结合。这样，结合平衡似乎是H + + hTF / 2N + X（2-）6 H-hTF / 2N（X），log K'= -log K-d + pH。磷酸化和硫酸化的蛋白质的δepsilon（246）与pH的关系曲线均为S型，pK（a）= 7.4。当协同阴离子与hTF / 2N一起使用时，丙二酸和草酸产生的pK（a）s为ca。 Δε（246）对pH的依赖性分别为6.9和7.1，但H207E突变蛋白的数值为7.3和7.6。为了将阴离子结合位点定位在hTF / 2N中，通过pH 7.4的差示紫外光谱法将硫酸盐与人转铁蛋白N瓣的单点突变体K296E，K296Q和K206Q结合。在K296E的情况下，硫酸盐结合产生的Delta epsilon（246）= 0，而对于K296Q，其结合的Delta epsilon（246）略为正。对于K206Q，结合产生的logK'为10.98，比硫酸盐与hTF / 2N野生型蛋白结合获得的常数小0.6个单位。这些数据表明，这两个赖氨酸残基在二价阴离子结合中具有重要作用。 [参考：27]

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