Date of Award

Spring 1985

Project Type


Program or Major


Degree Name

Doctor of Philosophy


Amino acids other than those that serve as ligands have been found to influence the chemical properties of transferrin iron. The catalytic ability of pyrophosphate to mediate transferrin iron release to a terminal acceptor is largely quenched by modification non-liganded histidine groups on the protein. The first order rate constants of iron release for several partially histidine modified protein samples were measured. A statistical method was employed to establish that one non-liganded histidine per metal binding domain was responsible for the reduction in rate constant. These results imply that the iron mediating chelator, pyrophosphate, binds directly to a histidine residue on the protein during the iron release process. EPR spectroscopic results are consistent with this interpretation. Kinetic and amino acid sequence studies of ovotransferrin and lactoferrin, in addition to human serum transferrin, have allowed the tentative assignment of His-207 in the N-terminal domain and His-535 in the C-terminal domain as the groups responsible for the reduction in rate of iron release.

The above concepts have been extended to lysine modified transferrin. Perchlorate induces changes in the EPR spectra and kinetics of iron release in human serum transferrin; similar effects are also induced by lysine modification and found to occur primarily in the C-terminal domain. Furthermore, the labilizing effect of 0.5 M sodium perchlorate on iron in the C-terminal site is largely quenched by lysine modification. The above experiments suggest that the well studied "perchlorate effects" in transferrin are caused by anion binding at a small number (<15) of lysines, probably located close to the metal.

Complexation of iron(III) to adenosine triphosphate (ATP) was also studied to gain insight into the nature of iron-ATP species present at physiological pH. At pH 7.0, mononuclear (metal:ligand < 1:3) and polynuclear (metal:ligand = 2:1 and 4:1) readily form in solution. The mononuclear complexes exhibit a g' = 4.3 EPR signal. ('31)P NMR spectra are observed when ATP is present in large excess. The polynuclear (4:1 metal:ligand) complex, although polydisperse in size, has a molecular weight greater than 50,000, indicating cluster formation (<250 iron atoms per cluster). These complexes are EPR silent and give no ('31)P NMR spectra consistent with findings for other reported polynuclear iron(III) complexes.