Chemical and immmunochemical characterization of the functional domains of hemopexin

Plasmin cleaves rabbit serum hemopexin (HPX; Mr 60000) into domain I which binds heme (Mr 35000) and domain II (Mr 25000) which may act in receptor binding. Modification with diethyl-pyrocarbonate of the 5 histidine (his) residues of domain I abolishes the normal spectrum of bound heme. Only 3 his of the heme-domain I complex were modified. These data indicate bis-histidyl coordination of heme in the domain as in intact HPX. Monoclonal antibodies were obtained by fusing mouse FOX NY myeloma cells with spleen cells from DBA mice immunized with rabbit heme-HPX. Of four stable hybridoma lines producing IgG/sub 1/, kappa type, JEN-1, -3 and-14 recognize the heme-binding domain I and JEN-11 recognizes domain II. Antibodies were bound to 12.5 nM mesoheme-/sup 125/I-HPX by incubating with 25 or 250 nM IgG before carrying out receptor binding studies with mouse Hepa cells. Only JEN-14 inhibited specific binding of heme-HPX to its receptor. JEN-11 had no effect on binding. Competitive inhibition experiments revealed that domains I and II interact with the receptor when presented to the cells as an equimolar complex with heme but not alone.