The binding of phosphate, bicarbonate, 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.(ABSTRACT TRUNCATED AT 250 WORDS)