SITE AND MECHANISM OF COVALENT BINDING OF 4-DIMETHYLAMINOPHENOL TO HUMAN-HEMOGLOBIN, AND ITS IMPLICATIONS TO THE FUNCTIONAL-PROPERTIES

  • 1 January 1983
    • journal article
    • research article
    • Vol. 24  (2) , 282-290
Abstract
4-Dimethylaminophenol, after i.v. injection, rapidly forms ferrihemoglobin and has been successfully used as an antidote 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 Hb. Hereby the physiological functions of human Hb are markedly altered. After binding of 2 molecules of 4-dimethylaminophenol to tetrameric Hb, the rate of autoxidation is increased about 6-fold. The O2 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 O2 affinity by 2,3-diphosphoglycerate is abolished, and the binding of 2,3-diphosphoglycerate to deoxyhemoglobin no longer functions. By molecular sieving, 2 alkylated H were separated: a Hb fraction with an unchanged low tetramer dimer dissociation, normal electronic spectra, and normal digestibility by carboxypeptidase A; and a 2nd 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. HbO2 evidently 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. The 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, Hb lacks allosteric transition upon ligation and is obviously frozen in its quaternary R-state.