title: Carbodiimide-mediated coupling of benzenepentacarboxylate to human hemoglobin: structural and functional consequences.
abstract: We have examined the covalent modification of HbA with BPC (benzenepentacarboxylate) whose carboxyl groups were activated with EDC [1-ethyl-3-(3-dimethyl- aminopropyl)-carbodiimide]. Reaction of deoxy-HbA at pH 8 with a 10-fold excess of BPC, preactivated with a 2-fold excess of EDC for 5 minutes, followed by anion-exchange chromatography, gives three components with p50 values of 1.15 (unreacted HbA), 11.7 and 7.6 mm of Hg at 20 degrees C (50 mM Bis-Tris pH 7.0). Component III does not dissociate into dimers upon dilution, but components I and II do. When deoxy-HbA is reacted at pH 6 with 10-fold BPC, preactivated with two-fold EDC for 5 minutes, the resultant HbA derivatives can be separated into three components, with p50 values at pH 7 of 14.2, 10.2 and 5.2 mm of Hg, respectively. All three components are stable tetramers. Oxygen binding by all of the covalent HbA-(BPC)x complexes is cooperative, pH sensitive, but IHP insensitive. The latter observation suggest that BPC is covalently bound to HbA's DPG/IHP binding site. This conclusion is corroborated by reversed phase HPLC analysis which shows that all five modified HbAs contain at least one modified beta chain. In addition, 4 of the 5 derivatives also contain modified alpha chains. No inter or intratetramer crosslinks are observed.
authors.affiliation: Duke University Marine Biomedical Center, Beaufort, NC 28516.
authors.forename: M
authors.initials: M
authors.lastname: Brouwer
authors.forename: R
authors.initials: R
authors.lastname: Cashon
authors.forename: J
authors.initials: J
authors.lastname: Bonaventura
mesh_terms: D001555:Benzene Derivatives; D001802:Blood Substitutes / Q000138:chemical synthesis* / Q000737:chemistry; D005022:Ethyldimethylaminopropyl Carbodiimide; D006441:Hemoglobin A / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015394:Molecular Structure; D010100:Oxygen
chemical_list: D001555:Benzene Derivatives; D001802:Blood Substitutes; D006441:Hemoglobin A; D005022:Ethyldimethylaminopropyl Carbodiimide; D010100:Oxygen