Thermodynamics of anion binding to human serum transferrin

Abstract

The binding of phosphate, biocarbonate, sulfate, and vanadate to human serum transferrin has been evaluated by two difference ultraviolet spectroscopic techniques. Direct titration of apotransferrin with bicarbonate, phosphate, and sulfate produces a strong negative absorbance near 245 nm, while titration with vanadate produces a positive absorbance in this region. Least-squares refinement of the absorbance data indicates that two anions of sulfate, phosphate, and vanadate bind to each transferrin molecule but that there is detectable binding of only a single bicarbonate anion. A second method used to study the thermodynamics of anion binding was competition equilibrium between anions for binding to the transferrin. The equilibrium constant for binding of the first equivalent of vanadate was determined by competition vs. phosphate and sulfate, while the equilibrium constant for binding of the second equivalent of bicarbonate was determined by competition vs. vanadate. Anion binding was described by two equilibrium constants for the successive binding of two anions per transferrin molecule: K1 = [A-Tr]/[A][Tr] and K2 = [A-Tr-A]/[A][A-Tr] where [A] represents the free anion concentration, [Tr] represents apotransferrin concentration, and [A-Tr] and [A-Tr-A] represent the concentrations of 1:1 and 2:1 anion-transferrin complexes, respectively. The results were the following: for phosphate, log K1 = 4.19 .+-. 0.03 and log K2 = 3.25 .+-. 0.21; for sulfate, log K1 = 3.62 .+-. 0.07 and log K2 = 2.79 .+-. 0.20; for vanadate, log K1 = 7.45 .+-. 0.10 and log K2 = 6.6 .+-. 0.30; for bicarbonate, log K1 = 2.66 .+-. 0.07 and log K2 = 1.8 .+-. 0.3. Previous studies have shown that vandate binds to the phenolic oxygens of the metal binding sites of transferrin [Harris W.R., and Carrano, C.J. (1984) J. Inorg. Biochem. 22, 201-218]. The direct competition observed between the other inorganic anions and vanadate and also the intense UV difference spectrum induced by binding of these anions to apotransferrin both indicate that the sulfate, phosphate, and bicarbonate are also interacting with these phenolic oxygens. Furthermore, binding of these anions is completely blocked in differic transferrin. A model is proposed that involves hydrogen bonding of the anions both to essential arginine and possibly lysine residues and to the phenolic hydrogens of the tyrosine residues of the metal binding site.