Plasmin cleaves rabbit serum apohemopexin (Mr = 60,000) at a single site producing a
heme-binding domain (I, Mr = 35,000) and a second domain (II, Mr = 25,000) (W. T. Morgan and A. Smith (1984) J. Biol. Chem. 259, 12001-12005). The absorbance spectra of
heme-domain I are indicative of a bis-histidyl coordination complex with the central
heme iron atom. Chemical modification of the 5
histidine residues of apo-domain I with
diethylpyrocarbonate abolished
heme binding, supporting this assignment. Upon binding
heme, domain I migrates more rapidly in
sucrose gradients, and, in sedimentation velocity experiments, the s value of domain I increases from 3.17 +/- 0.04 to 3.71 +/- 0.09, a notably large increase which indicates that the domain becomes much more compact. This conformational change which plays a pivotal role in
hemopexin function requires the bis-histidyl coordination with
heme iron and leads to a tighter association between domain I and domain II shown by the co-migration of
heme-domain I and domain II in
sucrose gradients. In turn, the association of
heme-domain I with domain II increases the thermal stability of the
heme-domain I chromophore. Results of binding studies using mouse
hepatoma cells and isolated domains indicate that domain I not only binds
heme but also plays a vital part in the
hemopexin-receptor interaction. The change in conformation of domain I upon
heme binding and the association between domains I and II induced by
heme are both notable determinants of the strength of the
hemopexin-receptor interaction, but an intact "hinge region" between the domains is not necessary for receptor binding. The importance of both domains in bringing about the transport function of
hemopexin is confirmed by the ability of three (two specific for domain I and one for domain II) of seven
monoclonal antibodies raised against
hemopexin to inhibit the
hemopexin-receptor interaction.