title: Site and mechanism of covalent binding of 4-dimethylaminophenol to human hemoglobin, and its implications to the functional properties.
abstract: 4-Dimethylaminophenol, after i.v. injection, rapidly forms ferrihemoglobin and has been successfully used in the treatment of cyanide poisoning. The catalytic ferrihemoglobin formation is terminated by thioether formation of oxidized 4-dimethylaminophenol with reduced glutathione or cysteine 93 beta of hemoglobin. Hereby the physiological functions of human hemoglobin are markedly altered. After binding of two molecules of 4-dimethylaminophenol to tetrameric hemoglobin, the rate of autoxidation is increased about 6-fold. The oxygen affinity is 10 times higher than normal, the Hill coefficient is diminished nearly to unity, and the Bohr effect is reduced by about 50%. The physiologically important allosteric regulation of the oxygen affinity by 2,3-diphosphoglycerate is abolished, and the binding of 2,3-diphosphoglycerate to deoxyhemoglobin no longer functions. By molecular sieving, two alkylated hemoglobins were separated: a hemoglobin fraction with an unchanged low tetramer dimer dissociation, normal electronic spectra, and normal digestibility by carboxypeptidase A; and a second fraction with a high degree of dissociation, altered electronic spectra, and impaired digestibility. A tryptic peptide was isolated containing cysteine 93 beta and histidine 146 beta cross-linked by an arylic compound missing the dimethylamine label. The following reaction mechanism is concluded: Oxy-hemoglobin catalyzes the oxidation of 4-dimethylaminophenol, and the oxidation product, presumably N,N-dimethylquinonimine, is bound covalently to cysteine 93 beta by a thioether linkage. This adduct is unstable and autoxidizes further with the liberation of dimethylamine. The resulting quinoid thioether electrophilically attacks the COOH-terminal histidine of the beta-chain, thereby forming an intramolecular cross-link. By this latter reaction, hemoglobin lacks allosteric transition upon ligation and is obviously frozen in its quaternary R-state.
mesh_terms: D000627:Aminophenols / Q000097:blood*; D000931:Antidotes; D001665:Binding Sites; D001667:Binding, Competitive; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D003486:Cyanides / Q000506:poisoning; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008706:Methemoglobin / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000097:blood; D010100:Oxygen / Q000097:blood; D011485:Protein Binding; D013438:Sulfhydryl Compounds / Q000097:blood
chemical_list: D000627:Aminophenols; D000931:Antidotes; D003486:Cyanides; D006454:Hemoglobins; D009943:Organophosphorus Compounds; D013438:Sulfhydryl Compounds; D008706:Methemoglobin; D010100:Oxygen; C020292:4-dimethylaminophenol