A key issue regarding the speciation of Al(3+) in serum is how well the ligands citric acid and phosphate can compete with the iron transport protein serum transferrin for the aluminum. Previous studies have attempted to measure binding constants for each ligand separately, but experimental problems make it very difficult to obtain stability constants with the accuracy required to make a meaningful comparison between these ligands. In this study, effective binding constants for Al-citrate and Al-phosphate at pH 7.4 have been determined using difference UV spectroscopy to monitor the direct competition between these ligands and transferrin. The analysis of this competition equilibrium also includes the binding of citrate and phosphate as anions to apotransferrin. The effective binding constants are 10(11.59) for the 1:1 Al-citrate complexes and 10(14.90) for the 1:2 Al-citrate complexes. The effective binding constant for the 1:2 Al-phosphate complex is 10(12.02). No 1:1 Al-phosphate complex was detected. Speciation calculations based on these effective binding constants indicate that, at serum concentrations of citrate and phosphate, citrate will be the primary low-molecular-mass ligand for aluminum. Formal stability constants for the Al-citrate system have also been determined by potentiometric methods. This equilibrium system is quite complex, and information from both electrospray mass spectrometry and difference UV experiments has been used to select the best model for fitting the potentiometric data. The mass spectra contain peaks that have been assigned to complexes having aluminum:citrate stoichiometries of 1:1, 1:2, 2:2, 2:3, and 3:3. The difference UV results were used to determine the stability constant for Al(H(-1)cta)-, which was then used in the least-squares fitting of the potentiometric data to determine stability constants for Al(Hcta)+, Al(cta), Al(cta)2(3-), Al(H(-1)cta)(cta)(4-), Al2(H(-1)cta)2(2-), and Al3(H(-1)cta)3(OH)(4-).

The exchange of Fe(3+), Tb(3+), In(3+), Ga(3+), and Al(3+) between the C-terminal metal-binding site of the serum iron transport protein transferrin and the low-molecular-mass serum chelating agent citrate has been studied at pH 7.4 and 25 degrees C. The removal of Ga(3+), In(3+), and Al(3+) follows simple saturation kinetics with respect to the citrate concentration. In contrast, removal of both Fe(3+) and Tb(3+) shows a combination of saturation and first-order kinetic behavior with respect to the citrate concentration. The saturation component is consistent with a mechanism for metal release in which access to the bound metal is controlled by a rate-limiting conformational change in the protein. The first-order kinetic pathway is very rapid for Tb(3+), and this is attributed to a direct attack of the citrate on the Tb(3+) ion within the closed protein conformation. It is suggested that this pathway is more readily available for Tb(3+) because of the larger coordination number for this cation and the presence of an aquated coordination site in the Tb(3+)-CO(3)-Tf ternary complex. There is relatively little variation in the k(max) values for the saturation pathway for Tb(3+), Ga(3+), Al(3+), and In(3+), but the k(max) value for Fe(3+) is significantly smaller. It is suggested that protein interactions across the interdomain cleft of transferrin largely control the release of the first group of metal ions, while the breaking of stronger metal-protein bonds slows the rate of iron release. The rates of metal binding to apotransferrin are clearly controlled in large part by the hydrolytic tendencies of the free metal ions. For the more amphoteric metal ions Al(3+) and Ga(3+), there is rapid protein binding, and the addition of citrate actually retards this reaction. In contrast, the nonamphoteric In(3+) ion binds very slowly in the absence of citrate, presumably due to the rapid formation of polymeric In-hydroxo complexes upon addition of the unchelated metal ion to the pH 7.4 protein solution. The addition of citrate to the reaction accelerates the binding of In(3+) to apoTf, presumably by forming soluble, mononuclear In-citrate complexes.