Use of iron-meso-tetra-(4-sulfonatophenyl)-porphine (FeTPPS) to examine hemopexin-mediated heme transport

Hemopexin (HPX) alters conformation upon binding heme as shown by circular dichroism (CD) while FeTPPS binds without changes in the CD spectrum . Therefore, FeTPPS was used to examine the importance of changes in HPX conformation for receptor binding and for HPX-mediated heme transport. FeTPPS-HPX binds to the HPX receptor on mouse hepatoma Hepa cells but with lower affinity then heme-HPX. Incubation of cells with 50 nM heme-/sup 125/I-HPX after 2.5 ..mu..M heme- or FeTPPS-HPX decreased binding from 0.34 pmol/mg protein to 0.10 and 0.27, respectively. Preincubation with 2.5 ..mu..M apoHPX reduced binding to the same extent as FeTPPS-HPX indicating that certain conformational changes in HPX increase the affinity of its receptor. Interestingly, FeTPPS-HPX inhibited heme uptake more effectively than heme-HPX. Preincubation of cells with 2.5 ..mu..M heme- or FeTPPS-HPX decreased /sup 55/Feheme uptake from /sup 55/Feheme-HPX (500 nM) by 28% and 70%, respectively; heme or FeTPPS alone had no effect. After incubation with 500 nM /sup 55/Feheme-HPX or /sup 55/FeTPPS-HPX for up to 30 minutes at 37/sup 0/C, /sup 55/Feheme was associated with the plasma-membrane and intracellular compartments but /sup 55/FeTPPs remained with the plasma membrane. FeTPPS presented to the cells as a complex with HPX inhibits HPX-mediated hememore » uptake by blocking events after heme-HPX binds to its receptor but needed for heme transport.« less