title: Fetal hemoglobin is much less prone to DNA cleavage compared to the adult protein.
abstract: Hemoglobin (Hb) is well protected inside the red blood cells (RBCs). Upon hemolysis and when free in circulation, Hb can be involved in a range of radical generating reactions and may thereby attack several different biomolecules. In this study, we have examined the potential damaging effects of cell-free Hb on plasmid DNA (pDNA). Hb induced cleavage of supercoiled pDNA (sc pDNA) which was proportional to the concentration of Hb applied. Almost 70% of sc pDNA was converted to open circular or linear DNA using 10µM of Hb in 12h. Hb can be present in several different forms. The oxy (HbO2) and met forms are most reactive, while the carboxy-protein shows only low hydrolytic activity. Hemoglobin A (HbA) could easily induce complete pDNA cleavage while fetal hemoglobin (HbF) was three-fold less reactive. By inserting, a redox active cysteine residue on the surface of the alpha chain of HbF by site-directed mutagenesis, the DNA cleavage reaction was enhanced by 82%. Reactive oxygen species were not directly involved in the reaction since addition of superoxide dismutase and catalase did not prevent pDNA cleavage. The reactivity of Hb with pDNA can rather be associated with the formation of protein based radicals.
authors.affiliation: Pure and Applied Biochemistry, Department of Chemistry, Lund University, Lund 22362, Sweden.
authors.forename: Sandeep
authors.initials: S
authors.lastname: Chakane
authors.affiliation: Pure and Applied Biochemistry, Department of Chemistry, Lund University, Lund 22362, Sweden.
authors.forename: Tiago
authors.initials: T
authors.lastname: Matos
authors.affiliation: Pure and Applied Biochemistry, Department of Chemistry, Lund University, Lund 22362, Sweden.
authors.forename: Karin
authors.initials: K
authors.lastname: Kettisen
authors.affiliation: Pure and Applied Biochemistry, Department of Chemistry, Lund University, Lund 22362, Sweden. Electronic address: leif.bulow@tbiokem.lth.se.
authors.forename: Leif
authors.initials: L
authors.lastname: Bulow
mesh_terms: D000328:Adult; D002474:Cell-Free System; D003545:Cysteine / Q000235:genetics; D004247:DNA / Q000737:chemistry*; D053837:DNA Cleavage; D005319:Fetal Hemoglobin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006441:Hemoglobin A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D017433:Protein Structure, Secondary; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D017382:Reactive Oxygen Species; D004247:DNA; D006441:Hemoglobin A; D005319:Fetal Hemoglobin; D003545:Cysteine
keywords: Adult hemoglobin; Cysteine; DNA cleavage; Fetal hemoglobin; Protein radicals; Supercoiled plasmid DNA