Browsing by Author "Alayash, Abdu I."

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    Alpha-hemoglobin stabilizing protein (AHSP) markedly decreases the redox potential and reactivity of alpha subunits of human HbA with hydrogen peroxide
    (The American Society for Biochemistry and Molecular Biology, Inc., 2012) Mollan, Todd L.; Banerjee, Sambuddha; Wu, Gang; Siburt, Claire J.Parker; Tsai, Ah-Lim; Olson, John S.; Weiss, Mitchell J.; Crumbliss, Alvin L.; Alayash, Abdu I.
    Background: AHSP modifies redox properties of bound α subunits. Results: Isolated hemoglobin subunits exhibit significantly different redox properties compared to HbA. A significant decrease in the reduction potential of α subunits bound to AHSP results in preferential binding of ferric α. Conclusion: AHSP:α subunit complexes do not participate in ferric-ferryl heme redox cycling. Significance: AHSP binding to α subunits inhibits subunit pseudoperoxidase activity.
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    Redox properties of human hemoglobin in complex with fractionated dimeric and polymeric human haptoglobin
    (Elsevier, 2014) Mollan, Todd L.; Jia, Yiping; Banerjee, Sambuddha; Wu, Gang; Kreulen, R.Timothy; Tsai, Ah-Lim; Olson, John S.; Crumbliss, Alvin L.; Alayash, Abdu I.
    Haptoglobin (Hp) is an abundant and conserved plasma glycoprotein, which binds acellular adult hemoglobin (Hb) dimers with high affinity and facilitates their rapid clearance from circulation after hemolysis. Humans possess three main phenotypes of Hp, designated Hp 1-1, Hp 2-1, and Hp 2-2. These variants exhibit diverse structural configurations and have been reported to be functionally nonequivalent. We have investigated the functional and redox properties of Hb–Hp complexes prepared using commercially fractionated Hp and found that all forms exhibit similar behavior. The rate of Hb dimer binding to Hp occurs with bimolecular rate constants of ~0.9 μM−1 s−1, irrespective of the type of Hp assayed. Although Hp binding does accelerate the observed rate of HbO2 autoxidation by dissociating Hb tetramers into dimers, the rate observed for these bound dimers is three- to fourfold slower than that of Hb dimers free in solution. Co-incubation of ferric Hb with any form of Hp inhibits heme loss to below detectable levels. Intrinsic redox potentials (E1/2) of the ferric/ferrous pair of each Hb–Hp complex are similar, varying from +54 to +59 mV (vs NHE), and are essentially the same as reported by us previously for Hb–Hp complexes prepared from unfractionated Hp. All Hb–Hp complexes generate similar high amounts of ferryl Hb after exposure to hydrogen peroxide. Electron paramagnetic resonance data indicate that the yields of protein-based radicals during this process are approximately 4 to 5% and are unaffected by the variant of Hp assayed. These data indicate that the Hp fractions examined are equivalent to one another with respect to Hb binding and associated stability and redox properties and that this result should be taken into account in the design of phenotype-specific Hp therapeutics aimed at countering Hb-mediated vascular disease.
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    Substitutions in the β subunits of sickle-cell hemoglobin improve oxidative stability and increase the delay time of sickle-cell fiber formation
    (American Society for Biochemistry and Molecular Biology, 2019) Meng, Fantao; Kassa, Tigist; Strader, Michael Brad; Soman, Jayashree; Olson, John S.; Alayash, Abdu I.
    After reacting with hydrogen peroxide (H2O2), sickle-cell hemoglobin (HbS, βE6V) remains longer in a highly oxidizing ferryl form (HbFe4+=O) and induces irreversible oxidation of “hot-spot” amino acids, including βCys-93. To control the damaging ferryl heme, here we constructed three HbS variants. The first contained a redox-active Tyr in β subunits (F41Y), a substitution present in Hb Mequon; the second contained the Asp (K82D) found in the β cleft of Hb Providence; and the third had both of these β 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 βF41Y and βF41Y/K82D enhanced ferryl Hb reduction by providing a pathway for electrons to reduce the heme via the Tyr-41 side chain. MS analysis of βCys-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 βK82D and βF41Y/K82D, respectively. Under hypoxia, βK82D and βF41Y/K82D HbS substitutions increased the delay time by ∼250 and 600 s before the onset of polymerization compared with the rHbS control and rHbS βF41Y, respectively. Moreover, at 60 °C, rHbS βK82D exhibited superior structural stability. Asp-82 also enhanced the function of Tyr as a redox-active amino acid in the rHbS βF41Y/K82D variant. We conclude that the βK82D and βF41Y substitutions add significant resistance to oxidative stress and anti-sickling properties to HbS and therefore could be potential genome-editing targets.