Reversible Changes in the Nem-Reactive —Sh Groups of Hemoglobin on Oxygenation-Deoxygenation

Morell, Samuel A.; Hoffman, P.; Ayers, Valborg E.; Taketa, F.

doi:10.1073/pnas.48.6.1057

Cited by 10 publications

(6 citation statements)

References 5 publications

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“…As previously observed (Taketa and Morell, 1969) and confirmed here for native hemoglobin, conversion of the cross-linked hemoglobins to the carbonyl derivative slightly decreases the reactivity with DTDP relative to that of the corresponding oxyhemoglobin derivatives. Furthermore, deoxygenation of the cross-linked hemoglobins decreased reactivity of the reactive thiol as previously observed for the native protein (Riggs, 1961;Morell et al, 1962;Benesch and Benesch, 1962). At alkaline pH (8.4) the rate constants for reaction of these proteins with DTDP is increased to the same degree (three-to fivefold relative to those at pH 7.0) as observed for native HbA 0 .…”

Section: Sulfhydryl Reactivitysupporting

confidence: 73%

Functional Comparison of Specifically Cross-Linked Hemoglobins Biased Toward the R and T States

Johnson

Adamson

Mauk

1998

Biophysical Journal

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Selected functional and spectroscopic properties of two human hemoglobin (HbA0) derivatives that were site-specifically cross-linked in the cleft between beta-chains where 2, 3-bisphosphoglycerate normally binds have been determined to assess the effects of the cross-linking on the behavior of the protein. Trimesoyl tris(3,5-dibromosalicylate) (TTDS) cross-links Hb between beta82Lys residues. The resulting TTDS-Hb exhibits a slower rate of oxygen dissociation and an increased rate of carbon monoxide association than observed for HbA0. The electron paramagnetic resonance (EPR) spectrum of TTDS-HbNO does not exhibit the hyperfine structure that is indicative of significant conformational change despite the fact that the 2,3-bisphosphoglycerate binding site is occupied by the cross-linking reagent. The reactivity of the beta93Cys residues of TTDS-Hb is only slightly decreased relative to that of HbA0. On the other hand, cross-linking Hb between Lys82 and the amino-terminal beta1Val group with trimesoyl tris(methyl phosphate) (TMMP) increases the rate of oxygen dissociation and reduces the rate of CO association relative to the rates observed for HbA0. In addition, the EPR spectrum of the TMMP-HbNO exhibits the three-line hyperfine structure that results from disruption of the proximal His-Fe bond of the alpha-chains, and the accessibility of the betaCys93 residues in this derivative is decreased fourfold. The present results are consistent with the conclusion that the quaternary structure of TTDS-Hb is shifted toward the R state whereas the quaternary structure of TMMP-Hb is shifted toward the T state and provides additional evidence that the identity of the residues involved in intramolecular cross-linking of hemoglobin within the 2,3-bisphosphoglycerate binding site between beta-chains can have a significant influence on the conformational and functional properties of the protein.

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Section: Sulfhydryl Reactivitysupporting

confidence: 73%

Functional Comparison of Specifically Cross-Linked Hemoglobins Biased Toward the R and T States

Johnson

Adamson

Mauk

1998

Biophysical Journal

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“…The high degree of B93C conservation in vertebrates [9], combined with its allosterically-controlled reactivity [13, 17] and high intracellular concentration suggest a prominent functional role in vivo. In the present work, we tested whether the β93Cys residue plays a role within the RBC antioxidant network.…”

Section: Discussionmentioning

confidence: 99%

“…This residue is conserved amongst vertebrates but its function remains unclear. Previous studies have shown that the reactivity of the β93Cys residues towards thiol-reactive agents is allosterically controlled by oxygen-dependent changes in hemoglobin conformation with the β93Cys in the R-state being more reactive towards nitrosating and alkylating agents [11–14] and to mercurials [15, 16] than T-state hemoglobin [17]. Moreover, the β93Cys can affect electron transfer reactions and limit ferrous heme-derived superoxide production and reactivity with other RBC components [18–21].…”

Section: Introductionmentioning

confidence: 99%

Antioxidant functions for the hemoglobin β93 cysteine residue in erythrocytes and in the vascular compartment in vivo

Vitturi

Sun

Harper

et al. 2013

Free Radical Biology and Medicine

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The β93 Cysteine (β93Cys) residue of hemoglobin is conserved in vertebrates but its function in the red blood cell (RBC) remains unclear. Since this residue is present at concentrations more than two orders of magnitude higher than enzymatic components of the RBC antioxidant network, a role in the scavenging of reactive species was hypothesized. Initial studies utilizing mice that express human hemoglobin with either Cys (B93C) or Ala (B93A) at the β93 positions, demonstrated that loss of the β93Cys did not affect activities nor expression of established components of the RBC antioxidant network (catalase, superoxide dismutase, peroxiredoxin-2, glutathione peroxidase, GSH:GSSG ratios). Interestingly, exogenous addition to RBC of reactive species that are involved in vascular inflammation demonstrated a role for the β93Cys in hydrogen peroxide and chloramine consumption. To simulate oxidative stress and inflammation in vivo, mice were challenged with LPS. Notably, LPS induced a greater degree of hypotension and lung injury in B93A versus B93C mice, which was associated with greater formation of RBC reactive species and accumulation of DMPO-reactive epitopes in the lung. These data suggest that the β93Cys is an important effector within the RBC antioxidant network contributing to the modulation of tissue injury during vascular inflammation.

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“…The quaternary conformation changes the environment of the βCys-93 residue on the proximal side of the haem. In the R state it is exposed to the solvent and reacts with sulphhydryl reagents [3,[42][43][44]. In the T state, solvent access to the cysteine is impeded by the salt bridges formed in deoxyhaemoglobin [3] and exogenous sulphhydryl reagents react much more slowly.…”

Section: Evidence Of T Statementioning

confidence: 99%

Cross-linking with O-raffinose lowers oxygen affinity and stabilizes haemoglobin in a non-cooperative T-state conformation

Jia

Ramasamy

Wood

et al. 2004

Biochemical Journal

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O-R-polyHbA(0) is an intra- and intermolecularly O-raffinose cross-linked derivative of deoxygenated human haemoglobin developed as an oxygen therapeutic. When compared with its native protein (HbA(0)), O-R-polyHbA(0) was found to be locked in the T (tense) quaternary conformation with a lower oxygen affinity, a reduced Bohr effect (50% of HbA(0)) and no measurable cooperativity (h=1). The kinetics of oxygen and CO binding to the protein indicate lower 'on' rates and faster 'off' rates than HbA(0) and the absence of effects of inositol hexaphosphate (IHP) on the kinetics. Other properties consistent with a T-like conformation are inaccessibility of the betaCys-93 thiol group of O-R-polyHbA(0), the hyperfine splitting from nitrogen in the EPR spectrum of the Fe(II)NO complex of O-R-polyHbA(0) and decreased flexibility in the distal haem pocket, as indicated by low-spin bis-histidine complexes detected by EPR of oxidized chains. A comparison of the properties of O-R-polyHbA(0) with those of HbA(0) with and without IHP, as well as the reaction of nitrite with deoxygenated haemoglobin, provide additional insights into the variations in the conformation of T-state haemoglobin in solution (modifications of the T state produced by adding organic phosphates, like IHP and 2,3-diphosphoglycerate). Although the physiological ramifications of locking HbA(0) in the T conformation with the O-raffinose are still unknown, valuable insights into haemoglobin function are provided by these studies of O-R-polyHbA(0).

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Reversible Changes in the Nem-Reactive —Sh Groups of Hemoglobin on Oxygenation-Deoxygenation

Cited by 10 publications

References 5 publications

Functional Comparison of Specifically Cross-Linked Hemoglobins Biased Toward the R and T States

Functional Comparison of Specifically Cross-Linked Hemoglobins Biased Toward the R and T States

Antioxidant functions for the hemoglobin β93 cysteine residue in erythrocytes and in the vascular compartment in vivo

Cross-linking with O-raffinose lowers oxygen affinity and stabilizes haemoglobin in a non-cooperative T-state conformation

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