The Penultimate Tyrosine Residues are Critical for the Genotoxic Effect of Human Hemoglobin.

ABSTRACT

Hemoglobin (Hb) is a potent oxidant outside the erythrocyte. The tyrosines α140 and ß145 play an important role in the structure and function of Hb by forming switch and hinge contacts. These carboxy-terminal residues of the alpha and beta chains, respectively, were replaced to phenylalanine and several different methods were used to characterize the obtained mutants including a comet and plasmid DNA cleavage assay. It was observed that the genotoxic effect was 40% higher for αY140F compared with the wildtype, the ßY145F and the double (αY140/ß145F) mutants as determined by the comet assay. Cleavage of purified plasmid DNA after Hb application also revealed that the αY140F mutant showed 2-fold higher activity, while the ßY145F and αY140/ß145F mutants reduced the activity compared to wildtype Hb. This study clearly indicates that the penultimate tyrosines are involved in the genotoxicity of Hb.