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 β145 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 βY145F and the double (αY140/β145F) 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 βY145F and αY140/β145F mutants reduced the activity compared to wildtype Hb. This study clearly indicates that the penultimate tyrosines are involved in the genotoxicity of Hb.

Pervasive Genomic Damage in Experimental Intracerebral Hemorrhage: Therapeutic Potential of a Mechanistic-Based Carbon Nanoparticle

“…Hb breakdown can form toxic products and generate reactive oxygen species (ROS) such as direct heme-derived superoxide (O 2 • – ), H 2 O 2 , and their products via secondary oxidative reactions of H 2 O 2 with Hb and ferrous and ferric Hb, thereby producing Fe­(IV)-ferrylHb and oxyferrylHb. Furthermore, these products can undergo additional oxidative reactions and are pro-inflammatory. ,, Finally, free Hb produces Hb–Hb dimers that readily enter cells and are oxidized to methemoglobin, which then reacts with H 2 O 2 to produce genotoxic oxyferryl Hb. − The amount of hemin liberated from hematoma during ICH may vary depending on hematoma size, ultimately affecting the severity and nature of hemin-induced neurotoxicity.…”

Fetal hemoglobin is much less prone to DNA cleavage compared to the adult protein

“…Hb can also induce DNA damages directly at the cellular level by rapid uptake and cleavage of available nucleic acids. This has been demonstrated for primary colon cells [16] , leukocytes [17] and lymphocytes [18] . The use of the comet assay has been instrumental for quantifying the genotoxic effects of Hb, which occur rapidly and already at low concentrations.…”

“…In addition, site-directed mutagenesis to β-Y145F has been made to characterize the redox property of the mutant. Replacement of this active tyrosine reduced the DNA cleavage activity [18] . Cysteine residues are also highly susceptible to redox modifications and particularly the irreversible oxidation of β-Cys93 have been emphasised [25] , [26] .…”