Covalent alteration of the prosthetic heme of human hemoglobin by BrCCl₃. Cross-linking of heme to cysteine residue 93

Recent studies have shown that a protein-bound heme adduct formed from the reaction of BrCCl₃ with myoglobin was due to bonding of the proximal histidine residue through the ring I vinyl of a heme-CCl₂ moiety. The present study reveals that BrCCl₃ also reacts with the heme of reduced human hemoglobin to form two protein-bound heme adducts. Edman degradation and mass spectrometry provided evidence that these protein-bound heme adducts were addition products in which heme-CCl₂ or heme-CCl₃ were bound to cysteine residue 93 of the β-chain of hemoglobin. It appeared that the cysteine residue was bonded regiospecifically to the ring I vinyl group of the altered heme moiety, because the nonprotein-bound products of the reaction included the β-carboxyvinyl and α-hydroxy-β-trichloromethylethyl derivatives of the ring I vinyl moiety of heme. The absorption spectra of the protein-bound adducts in both the oxidized and reduced states were highly similar to those described for hemichromes, which are thought to be involved in the formation of Heinz bodies and subsequent red cell lysis.