CALORIMETRIC STUDIES OF THE BINDING OF FERRIC IONS TO HUMAN SERUM TRANSFERRIN

The binding of ferric ions, chelated with nitrilotriacetate, to human serum transferrin (hTF) has been studied using ultrasensitive titratio n calorimetry. Studies were done in both the presence and the absence of the synergistic bicarbonate anion. It was found that the C-site of hTF is capable of weakly binding bicarbonate (K of 250 M-1, DELTAH of -8 kcal) at the binding site even before ferric ion is added, although this does not happen to the same extent at the N-site. When preinsert ion of the bicarbonate ion occurs, then ferric ion can subsequently bi nd very quickly to the C-site. Although the chelated ferric ion can bi nd weakly to the N-site in a fast reaction, the insertion of the bicar bonate ion occurs subsequently in a slow endothermic reaction. Binding of ferric ion to both sites is quickly reversible in the absence of b icarbonate but becomes kinetically controlled for long periods of time once bicarbonate has inserted into the metal-binding site due to the long time required for release of ferric ion. Estimates of the heats o f binding to each site, apparent binding constants, and heat capacitie s of binding are made for different sets of solution conditions. Resul ts from this study are compared to earlier results with ovotransferrin (Lin, L.-N., Mason, A. B., Woodworth, R. C., & Brandts, J. F. (1991) Biochemistry 30, 11660-11669), with major differences and some similar ities noted.