Date of Award
Program or Major
Doctor of Philosophy
N Dennis Chasteen
The role of non-coordinated histidines in the iron release mechanism of human serum transferrin has been investigated by chemical modification of the protein with ethoxyformic anhydride and aquopentaamine ruthenium(II), reagents specific for histidines under mild conditions. Kinetic studies of the iron release reaction at pH 5 under the action of different mediators e.g., PPi, Pi, Cit, ATP, GTP, and DPG, show that ethoxyformic anhydride imparts increased stability to the protein with rate constants for the C-terminal site decreased by factors from 2 to 10. The largest effects were seen with intracellular iron chelators GTP and DPG. A protonated imidazole group near the metal perhaps serves as a binding site for the triphosphate chelators. The binding of such chelators to a nearby histidine may assist in the formation of a quaternary intermediate of the type Chel-Fe-Transferrin-HCO$\sb3$ prior to release of iron.
Modification using aquopentaamine ruthenium(II) a reagent for surface accessible histidine residues, enhances the rate of release from the N-terminal site but has no effect the C-terminal site. The Tsou Chen-Lu statistical method used to analyze the rate data suggests the involvement of two histidines in the N-terminal lobe not conserved in the C-terminal lobe. These results may explain the kinetic liability of the N-terminal site relative to the C-terminal site in acidic solutions.
The distance between the metal site and nearby histidines was calculated from fluorescence energy transfer measurements using Tb(III) in the iron(III) binding site as the donor and pentaamine ruthenium(III) modified histidines as the acceptor chromophore. The fluorescence measurements imply two histidines in each lobe are responsible for quenching of the Tb(III) emission. Using upper and lower limits for the index of refraction and quantum yield and assuming the energy transfer follows parallel first order kinetics, a donor-acceptor distance between 1.32 nm and 1.42 nm was obtained.
Martin, Donna M., "The role of histidine in the mechanism of iron release from human serum transferrin" (1988). Doctoral Dissertations. 1563.