Calorimetric studies of melanotransferrin (p97) and its interaction with iron.
The mammalian molecule melanotransferrin (mTf), also called p97, is a member of the transferrin family of molecules. It exists in both secreted and glycosylphosphatidylinositol-anchored forms and is thought to play a role in angiogenesis and in transporting iron across the blood brain barrier. The binding affinity of iron to this molecule has not been formally established. Here, the binding of ferric ion (chelated with a 2-fold molar ratio of nitrilotriacetate) to mTf has been studied using isothermal titration calorimetry and differential scanning calorimetry. One iron-binding site was determined for mTf with similar binding characteristics to other transferrins. In the absence of bicarbonate, binding occurs quickly with an apparent association constant of 2.6 x 10(7) M(-1) at 25 degrees C. The presence of bicarbonate introduces kinetic effects that prevent direct determination of the apparent binding constant by isothermal titration calorimetry. Differential scanning calorimetry thermograms of mTf unfolding in the presence and absence of iron were therefore used to determine the apparent binding constant in the bicarbonate-containing system; at pH 7.5 and 25 degrees C, iron binding occurs in a 1:1 ratio with a K(app) of 4.4 x 10(17) M(-1). This affinity is intermediate between the high and low affinity lobes of transferrin and suggests that mTf is likely to play a significant role in iron transport where the high affinity lobe of transferrin is occupied or where transferrin is in proportionally low concentrations.
Creagh
A Louise
AL
Michael Smith Laboratories and Biomedical Research Centre, Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada.
Tiong
Jacqueline W C
JW
Tian
Mei Mei
MM
Haynes
Charles A
CA
Jefferies
Wilfred A
WA
D000818:Animals; D000951:Antigens, Neoplasm; D001639:Bicarbonates; D002152:Calorimetry, Differential Scanning; D006224:Cricetinae; D007501:Iron / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D058950:Melanoma-Specific Antigens; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors
D000951:Antigens, Neoplasm; D001639:Bicarbonates; D058950:Melanoma-Specific Antigens; D009363:Neoplasm Proteins; D007501:Iron