PH-DEPENDENCE OF SPECIFIC DIVALENT ANION-BINDING TO THE N-LOBE OF RECOMBINANT HUMAN TRANSFERRIN

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.