Determination of extinction coefficients of human hemoglobin in various redox states

Meng, Fantao; Alayash, Abdu I.

doi:10.1016/j.ab.2017.01.002

Cited by 89 publications

(113 citation statements)

References 48 publications

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“…The α and β globin chains, and the heme moieties of HbS were separated into three distinct peaks, similar to early reported HbA and HbS chromatographic profiles (10) (as shown in the first two panels of Figure 6). The heme peaks for HbA, TD-1 treated and non-treated HbS all eluted at 16.7 minutes (not shown).…”

Section: Resultssupporting

confidence: 86%

“…The kinetic results reported here were derived from temperature-jump experiments measured during light scattering (at 700 nm) which reflects Hb polymerization. For each experiment spectrophotometric analysis was used (at time zero) to confirm complete deoxygenation of Hb (10). The increase in turbidity (at 700 nm) at various time points (for each protein concentration) was plotted as a function of time to generate typical sigmoidal curves shown in Figure 2A as previously reported; the polymerization delay time (td) decreased as the protein concentration increased and was further prolonged upon Hb dilution.…”

Section: Resultsmentioning

confidence: 99%

“…Wild type hemoglobin (HbA) used in this study was prepared as previously described (10). Sickle cell Hb (HbS) was purchased from Sigma Aldrich (St. Louise, Missouri, US) and its purity was verified by isoelectric focusing and reversed phase-HPLC.…”

Section: Methodsmentioning

confidence: 99%

“…Sickle cell Hb (HbS) was purchased from Sigma Aldrich (St. Louise, Missouri, US) and its purity was verified by isoelectric focusing and reversed phase-HPLC. Hb concentrations were determined spectrophotometrically and were based on ε = 16.61 mM −1 cm −1 at 576 nm for oxy (HbO 2 ), 3.78 mM −1 cm −1 at 630 nm for ferric (met) Hb per heme with a molecular weight of 16,000 g/mol of heme (10). Unless otherwise stated, the Hb concentration was given as heme equivalents and the final compositions of the solvent for all measurements were carried out in potassium phosphate buffer (50 mM of phosphate, pH 7.4) at 22 °C.…”

Section: Methodsmentioning

confidence: 99%

“…H 2 O 2 -induced ferryl Hb species formation was evaluated following our previously-described method (10). First, TD-1 (60 μM) was mixed with HbS (60 μM) in a cuvette and incubated for 15 min at 37 °C.…”

Section: Methodsmentioning

confidence: 99%

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Targeting βCys93 in hemoglobin S with an antisickling agent possessing dual allosteric and antioxidant effects

et al. 2017

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Sickle cell disease (SCD) is an inherited blood disorder caused by a β globin gene mutation of hemoglobin (HbS). The polymerization of deoxyHbS and its subsequent aggregation (into long fibers) is the primary molecular event which leads to red blood cell (RBC) sickling and ultimately hemolytic anemia. We have recently suggested that HbS oxidative toxicity may also contribute to SCD pathophysiology due to its defective pseudoperoxidase activity. As a consequence, a persistently higher oxidized ferryl heme is formed which irreversibly oxidizes “hotspot” residues (particularly βCys93) causing protein unfolding and subsequent heme loss. In this report we confirmed first, the allosteric effect of a newly developed reagent (Di(5-(2,3-dihydro-1,4-benzodioxin-2-yl)-4H-1,2,4-triazol-3-yl)disulfide (TD-1) on oxygen affinity within SS RBCs. There was a considerable left shift in oxygen equilibrium curves (OECs) representing treated SS cells. Under hypoxic conditions, TD-1 treatment of HbS resulted in an approximately 200 sec increase in the delay time of HbS polymerization over the untreated HbS control. The effect of TD-1 binding to HbS was also tested on oxidative reactions by incrementally treating HbS with increasing hydrogen peroxide (H2O2) concentrations. Under these experimental conditions, ferryl levels were consistently reduced by approximately 35% in the presence of TD-1. Mass spectrometric analysis confirmed that upon binding to βCys93, TD-1 effectively blocked irreversible oxidation of this residue. In conclusion, TD-1 appears to shield βCys93 (the end point of radical formation in HbS) and when coupled with its allosteric effect on oxygen affinity may provide new therapeutic modalities for the treatment of SCD.

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Section: Resultssupporting

confidence: 86%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Targeting βCys93 in hemoglobin S with an antisickling agent possessing dual allosteric and antioxidant effects

et al. 2017

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Caffeic acid: an antioxidant with novel antisickling properties

et al. 2021

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It is well documented that caffeic acid (3,4‐dihydroxycinnamic acid) (CA) interacts with and inhibits the oxidative reactions of myoglobin (Mb) and hemoglobin (Hb), and this interaction underlies its antioxidative action in meat. Sickle cell hemoglobin (HbS) is known for its tendency to oxidize more readily than normal HbA in the presence of hydrogen peroxide (H2O2), which leads to a more persistent and highly oxidizing ferryl Hb (HbFe4+). We have investigated the effects of CA on HbS oxidation intermediates, specifically on the ferric/ferryl forms. At a low concentration of H2O2 (0.5‐fold over heme), we observed a fivefold reduction in the amount of HbFe4+ accumulated in a mixture of ferric and H2O2 solution. Higher levels of H2O2 (onefold and twofold over heme) led to a lesser threefold and twofold reduction in the content of HbFe4+, respectively, possibly due to the saturation of the binding sites on the Hb molecule. The most intriguing finding was that when 5‐molar excess CA over heme was used, and a considerable increase in the delay time of HbS polymerization to approximately 200 s was observed. This delay in polymerization of HbS is theoretically sufficient to avoid microcapillary blockage and prevent vasoconstrictions in vivo. Mass spectrometry analysis indicated that CA was more extensively covalently bonded to βCys93 than to βCys112 and αCys104. The dual antioxidant and antisickling properties of CA may be explored further to maximize its therapeutic potential in SCD.

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