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 10 µM of Hb in 12 h. 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.

Site-directed mutagenesis of cysteine residues alters oxidative stability of fetal hemoglobin

“…However, electron transfer can be mediated via other residues. For instance, the more distant α19 can interact via αTyr24, located 16.5 Å from the α-heme iron and 7.9 Å from α19, as proposed previously as a route of electron transfer [22] .…”

“…Cell-free Hb is used as the principal component but the oxidative reactions of the protein present problems that must be resolved. In this study, the focus has been on protein engineering of HbF as a substitute for HbA due to its reported potential advantages as a starting material for oxygen therapeutic development [12] , [21] , [22] . However, even though αA19C appears to be a promising mutation in terms of providing oxidative stability of HbF, there could also be other possible positions where cysteine residues can be introduced in the protein.…”

“…The gene expressing wildtype recombinant HbF (rHbF) was previously inserted into the pETDuet-1 plasmid from Novagen [21] . Site-directed mutagenesis was used to mutate the rHbF gene at specific sites of interest, and the method has been described previously [22] . Three mutants were formed by; 1) substitution of an alanine residue for cysteine at position 19 in the α-chain (αA19C); 2) substitution of a cysteine residue for alanine at position 93 in the γ-chain (γC93A); and 3) a mutant with both previous substitutions (αA19C/γC93A).…”

“…The E. coli containing rHbF, αA19C, γC93A or αA19C/γC93A were all cultivated in the same manner and the expression and purification procedures were based on previous protocols [21] , [22] , [23] , with some modifications. 500 ml Terrific Broth media in 2 l Erlenmeyer flasks were inoculated with overnight starter culture and immediately induced with 0.1 mM isopropyl β- D -1-thiogalactopyranoside (IPTG, Saveen & Werner) and supplemented with 0.24 mM δ-aminolevulinic acid (Sigma-Aldrich).…”

Umbilical cord blood as a source for red‐blood‐cell transfusion in neonatology: a systematic review

“…The lower oxygen affinity of adult Hb does not seem to influence fetal metabolism when Hb levels are normal; nevertheless, during moderate or severe anaemia, it has been shown that cord blood samples containing higher levels of adult Hb have also a lower pH and higher base deficit . Notably, as compared to adult Hb, fetal Hb has a significantly greater capacity to generate nitric oxide (which plays an important role in regulating vascular tone and blood flow) , is oxidatively more stable and is less prone to the DNA cleavage activity .…”