EQUILIBRIUM-CONSTANTS FOR THE BINDING OF INDIUM(III) TO HUMAN SERUM TRANSFERRIN

Equilibrium constants have been determined for the binding of In3+ to the two specific metal-binding sites of human serum transferrin. Nitrilotriacetic acid (NTA) was used as a competitive low molecular weight chelating agent. Prior to conducting the protein studies, a new set of equilibrium constants describing the indium NTA system were determined by a combination of potentiometric and spectrophotometric techniques. The indium-NTA system is described by three equilibrium constants: log beta(110) = 13.81 +/- 0.05, log beta(120) = 23.70 +/- 0.09, and log beta(121) = 26.57 +/- 0.07. Indium binding constants for transferrin were measured by difference ultraviolet spectroscopy at 25 degrees C in pH 7.4 solutions of 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid which also contained 5 mM sodium bicarbonate. The observed binding constants are log K-1* = 18.52 +/- 0.16 and log K-2* = 16.64 +/- 0.50. These have been corrected to carbonate-independent metal binding constants of log K-1M = 18.74 and log K-2M = 16.86. These constants are substantially smaller than previously reported values for the In-transferrin binding constants and are smaller than the transferrin binding constants for either Ga3+ or Fe3+. However, when hydrolysis of the free metal ions is taken into account, the more extensive hydrolysis df the Ga3+ ion at pH 7.4 leads to a reversal in stability such that In3+ is bound more strongly to transferrin at physiological pH. Linear free energy relationships (LFER) for the complexation of Fe3+ and In3+ were constructed to evaluate the consistency between the transferrin results and the stability constants for Fe3+ and In3+ with low molecular weight (LMW) ligands. However, the linear free energy relationships between Fe3+ and In3+ show unusual differences among different types of low molecular weight ligands, and there is no conclusive fit of the In-transferrin binding constants to the LMW LFER.