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RSS FeedsSubstitutions in the {beta} subunits of sickle-cell hemoglobin improve oxidative stability and increase the delay time of sickle-cell fiber formation [Protein Structure and Folding] (Journal of Biological Chemistry)

 
 

15 march 2019 09:02:32

 
Substitutions in the {beta} subunits of sickle-cell hemoglobin improve oxidative stability and increase the delay time of sickle-cell fiber formation [Protein Structure and Folding] (Journal of Biological Chemistry)
 




After reacting with hydrogen peroxide (H2O2), sickle-cell hemoglobin (HbS, ?E6V) remains longer in a highly oxidizing ferryl form (HbFe4+=O) and induces irreversible oxidation of `hot-spot` amino acids, including ?Cys-93. To control the damaging ferryl heme, here we constructed three HbS variants. The first contained a redox-active Tyr in ? subunits (F41Y), a substitution present in Hb Mequon; the second contained the Asp (K82D) found in the ? cleft of Hb Providence; and the third had both of these ? substitutions. Both the single Tyr-41 and Asp-82 constructs lowered the oxygen affinity of HbS but had little or no effects on autoxidation or heme loss kinetics. In the presence of H2O2, both rHbS ?F41Y and ?F41Y/K82D enhanced ferryl Hb reduction by providing a pathway for electrons to reduce the heme via the Tyr-41 side chain. MS analysis of ?Cys-93 revealed moderate inhibition of thiol oxidation in the HbS single F41Y variant and dramatic 3- to 8-fold inhibition of cysteic acid formation in rHbS ?K82D and ?F41Y/K82D, respectively. Under hypoxia, ?K82D and ?F41Y/K82D HbS substitutions increased the delay time by ~250 and 600 s before the onset of polymerization compared with the rHbS control and rHbS ?F41Y, respectively. Moreover, at 60 C, rHbS ?K82D exhibited superior structural stability. Asp-82 also enhanced the function of Tyr as a redox-active amino acid in the rHbS ?F41Y/K82D variant. We conclude that the ?K82D and ?F41Y substitutions add significant resistance to oxidative stress and anti-sickling properties to HbS and therefore could be potential genome-editing targets.


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37 viewsCategory: Biochemistry
 
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