We have determined the complete primary structure of human hemopexin, a plasma beta-glycoprotein that specifically binds one heme with high affinity and transports it to hepatocytes for salvage of the iron. Human hemopexin (Mr approximately equal to 63,000) consists of a single polypeptide chain containing 439 amino acid residues with six intrachain disulfide bridges. The amino-terminal threonine residue is blocked by an O-linked galactosamine oligosaccharide, and the protein has five glucosamine oligosaccharides N-linked to the acceptor sequence Asn-X-Ser/Thr. The 18 tryptophan residues are arranged in four clusters, and 12 of the tryptophans are conserved in homologous positions. Computer-assisted analysis of the internal homology in amino acid sequence indicates that hemopexin consists of two similar halves, thus suggesting duplication of an ancestral gene. Limited tryptic digestion cleaves apohemopexin after arginine-216 into two half-molecules, whereas heme-saturated hemopexin is cleaved after lysine-101. The half-molecules are connected by a histidine-rich hinge-like region that contains two glucosamine oligosaccharides. A structural model for human hemopexin is proposed that is based on these properties and on computer-assisted predictions of the secondary structure and the hydrophilic/hydrophobic character. In this model alpha-helices and beta-turns predominate, and the two halves are connected by an exposed connecting region in apohemopexin that becomes inaccessible to trypsin in hemesaturated hemopexin. Many segments of hemopexin are similar to sequences of other heme proteins, but no overall structural relationship of hemopexin to any other heme protein was identified.

The feline leukemia virus subgroup C receptor (FLVCR) is a heme export protein that is required for proerythroblast survival and facilitates macrophage heme iron recycling. However, its mechanism of heme export and substrate specificity are uncharacterized. Using [(55)Fe]heme and the fluorescent heme analog zinc mesoporphyrin, we investigated whether export by FLVCR depends on the availability and avidity of extracellular heme-binding proteins. Export was 100-fold more efficient when the medium contained hemopexin (K(d) < 1 pm) compared with albumin (K(d) = 5 nm) at the same concentration and was not detectable when the medium lacked heme-binding proteins. Besides heme, FLVCR could export other cyclic planar porphyrins, such as protoporphyrin IX and coproporphyrin. However, FLVCR has a narrow substrate range because unconjugated bilirubin, the primary breakdown product of heme, was not transported. As neither protoporphyrin IX nor coproporphyrin export improved with extracellular hemopexin (versus albumin), our observations further suggest that hemopexin, an abundant protein with a serum concentration (6.7-25 mum) equivalent to that of the iron transport protein transferrin (22-31 mum), by accepting heme from FLVCR and targeting it to the liver, might regulate macrophage heme export and heme iron recycling in vivo. Final studies show that hemopexin directly interacts with FLVCR, which also helps explain why FLVCR, in contrast to some major facilitator superfamily members, does not function as a bidirectional gradient-dependent transporter. Together, these data argue that hemopexin has a role in assuring systemic iron balance during homeostasis in addition to its established role as a scavenger during internal bleeding or hemolysis.

We have determined the complete primary structure of human hemopexin, a plasma beta-glycoprotein that specifically binds one heme with high affinity and transports it to hepatocytes for salvage of the iron. Human hemopexin (Mr approximately equal to 63,000) consists of a single polypeptide chain containing 439 amino acid residues with six intrachain disulfide bridges. The amino-terminal threonine residue is blocked by an O-linked galactosamine oligosaccharide, and the protein has five glucosamine oligosaccharides N-linked to the acceptor sequence Asn-X-Ser/Thr. The 18 tryptophan residues are arranged in four clusters, and 12 of the tryptophans are conserved in homologous positions. Computer-assisted analysis of the internal homology in amino acid sequence indicates that hemopexin consists of two similar halves, thus suggesting duplication of an ancestral gene. Limited tryptic digestion cleaves apohemopexin after arginine-216 into two half-molecules, whereas heme-saturated hemopexin is cleaved after lysine-101. The half-molecules are connected by a histidine-rich hinge-like region that contains two glucosamine oligosaccharides. A structural model for human hemopexin is proposed that is based on these properties and on computer-assisted predictions of the secondary structure and the hydrophilic/hydrophobic character. In this model alpha-helices and beta-turns predominate, and the two halves are connected by an exposed connecting region in apohemopexin that becomes inaccessible to trypsin in hemesaturated hemopexin. Many segments of hemopexin are similar to sequences of other heme proteins, but no overall structural relationship of hemopexin to any other heme protein was identified.

We have determined the complete primary structure of human hemopexin, a plasma beta-glycoprotein that specifically binds one heme with high affinity and transports it to hepatocytes for salvage of the iron. Human hemopexin (Mr approximately equal to 63,000) consists of a single polypeptide chain containing 439 amino acid residues with six intrachain disulfide bridges. The amino-terminal threonine residue is blocked by an O-linked galactosamine oligosaccharide, and the protein has five glucosamine oligosaccharides N-linked to the acceptor sequence Asn-X-Ser/Thr. The 18 tryptophan residues are arranged in four clusters, and 12 of the tryptophans are conserved in homologous positions. Computer-assisted analysis of the internal homology in amino acid sequence indicates that hemopexin consists of two similar halves, thus suggesting duplication of an ancestral gene. Limited tryptic digestion cleaves apohemopexin after arginine-216 into two half-molecules, whereas heme-saturated hemopexin is cleaved after lysine-101. The half-molecules are connected by a histidine-rich hinge-like region that contains two glucosamine oligosaccharides. A structural model for human hemopexin is proposed that is based on these properties and on computer-assisted predictions of the secondary structure and the hydrophilic/hydrophobic character. In this model alpha-helices and beta-turns predominate, and the two halves are connected by an exposed connecting region in apohemopexin that becomes inaccessible to trypsin in hemesaturated hemopexin. Many segments of hemopexin are similar to sequences of other heme proteins, but no overall structural relationship of hemopexin to any other heme protein was identified.