Domain structure of rabbit hemopexin. Isolation and characterization of a heme-binding glycopeptide.
Plasmin preferentially cleaves rabbit hemopexin at a single site, generating two nondisulfide-linked carbohydrate-containing fragments. In contrast, heme-hemopexin is almost totally resistant to this enzyme and is more resistant than the apoprotein to digestion by trypsin, chymotrypsin, papain, subtilisin, and proteinase K as well. Plasmin digestion dramatically shortens the plasma clearance time of the molecule. The larger glycopeptide (I), shown to be derived from the amino terminus of the parent molecule by sequence analysis, has a molecular weight near 35,000 with a pI of 5.0. It binds 1 mol of heme per mol in a manner analogous to intact hemopexin, molecular weight near 60,000 and pI 5.8. The smaller glycopeptide (II) has a molecular weight near 25,000, a pI of 6.4, and does not bind heme. Of the four oligosaccharides of rabbit hemopexin, peptide I contains three oligosaccharides and peptide II contains one. At micromolar concentrations, the two peptides migrate together during centrifugation through sucrose gradients in the presence, but not in the absence, of heme. Peptide I has a far UV circular dichroism spectrum indicating it has some alpha-helical and extensive nonrepeating peptide structures whereas peptide II appears to be almost exclusively in a beta-sheet conformation. Peptide II is responsible for most of the positive ellipticity at 231 nm of native apohemopexin, but the increase in ellipticity at 231 nm characteristic of heme-hemopexin is not seen when peptide I binds heme, even in the presence of peptide II.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002499:Centrifugation, Density Gradient; D002942:Circular Dichroism; D005341:Fibrinolysin / Q000378:metabolism; D006020:Glycopeptides / Q000302:isolation & purification* / Q000378:metabolism; D006418:Heme / Q000378:metabolism*; D006466:Hemopexin / Q000032:analysis*; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D000596:Amino Acids; D006020:Glycopeptides; D006418:Heme; D006466:Hemopexin; D005341:Fibrinolysin