title: Reversible binding of heparin to the loop peptide of endotoxin neutralizing protein.
abstract: Endotoxin neutralizing protein (ENP) from Limulus polyphemus is an amphipathic, 11.8 kDa protein with an isoelectric point of 10.2. ENP neutralizes lipopolysaccharide (LPS) and possesses antibacterial activity against Gram-negative bacteria. Heparin binds to ENP and blocks its LPS-neutralizing activity. The relative blocking activity of heparin is equal to low molecular weight heparin and polyanetholsulfonic acid > heparan sulfate > chondroitin sulfate A > chondroitin sulfate C. Endoproteinase Glu-C hydrolysis of recombinant ENP results in four major peptides, three of which are seen following separation on reversed phase HPLC. Heparin binds to the loop peptide (31-72), which includes the heparin binding consensus sequence XBBXBX between the two cysteine residues of ENP. When heparin is added to the digest and then applied to a C18 column, the loop peptide is bound; however, it dissociates and elutes with either 5 M NaCl or 0.1 M sodium phosphate, demonstrating reversible binding to heparin. LPS and lipid A both bind to the loop peptide and remove it from digests of ENP; however, neither complex could be dissociated by salt or sodium phosphate. Heparin, LPS, and lipid A individually bind to the same site on ENP.
authors.affiliation: Associates of Cape Cod, Inc., Falmouth, MA 02540, USA.
authors.forename: R J
authors.initials: RJ
authors.lastname: Ridge
authors.forename: E J
authors.initials: EJ
authors.lastname: Paus
authors.forename: T J
authors.initials: TJ
authors.lastname: Novitsky
authors.forename: P A
authors.initials: PA
authors.lastname: Ketchum
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D023181:Antimicrobial Cationic Peptides; D060829:Arthropod Proteins; D001665:Binding Sites; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D006493:Heparin / Q000378:metabolism*; D007447:Invertebrate Hormones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000378:metabolism; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*
chemical_list: D023181:Antimicrobial Cationic Peptides; D060829:Arthropod Proteins; D007447:Invertebrate Hormones; D008070:Lipopolysaccharides; D010455:Peptides; C034705:antilipopolysaccharide factor (Limulus); D006493:Heparin