The role of β93 Cys in the inhibition of Hb S fiber formation

Kelly M. Knee, Catherine K. Roden, Mark Flory, Ishita Mukerji

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Recent studies have suggested that nitric oxide (NO) binding to hemoglobin (Hb) may lead to the inhibition of sickle cell fiber formation and the dissolution of sickle cell fibers. NO can react with Hb in at least 3 ways: 1) formation of Hb(II)NO, 2) formation of methemoglobin, and 3) formation of S-nitrosohemoglobin, through nitrosylation of the β93 Cys residue. In this study, the role of β93 Cys in the mechanism of sickle cell fiber inhibition is investigated through chemical modification with N-ethylmaleimide. UV resonance Raman, FT-IR and electrospray ionization mass spectroscopic methods in conjunction with equilibrium solubility and kinetic studies are used to characterize the effect of β93 Cys modification on Hb S fiber formation. Both FT-IR spectroscopy and electrospray mass spectrometry results demonstrate that modification can occur at both the β93 and α104 Cys residues under relatively mild reaction conditions. Equilibrium solubility measurements reveal that singly-modified Hb at the β93 position leads to increased amounts of fiber formation relative to unmodified or doubly-modified Hb S. Kinetic studies confirm that modification of only the β93 residue leads to a faster onset of polymerization. UV resonance Raman results indicate that modification of the α104 residue in addition to the β93 residue significantly perturbs the α1β2 interface, while modification of only β93 does not. These results in conjunction with the equilibrium solubility and kinetic measurements are suggestive that modification of the α104 Cys residue and not the β93 Cys residue leads to T-state destabilization and inhibition of fiber formation. These findings have implications for understanding the mechanism of NO binding to Hb and NO inhibition of Hb S fiber formation.

Original languageEnglish (US)
Pages (from-to)181-193
Number of pages13
JournalBiophysical Chemistry
Volume127
Issue number3
DOIs
StatePublished - May 1 2007
Externally publishedYes

Fingerprint

Sickle Hemoglobin
hemoglobin
Solubility
Nitric Oxide
Hemoglobins
nitric oxide
fibers
Fibers
Methemoglobin
Ethylmaleimide
Polymerization
solubility
Mass Spectrometry
Spectrum Analysis
Kinetics
kinetics
cells
Electrospray ionization
Chemical modification
destabilization

Keywords

  • Fiber formation
  • Hemoglobin
  • NO
  • S-nitrosylhemoglobin
  • Sickle cell disease
  • Thiol modification
  • UV resonance Raman

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Physical and Theoretical Chemistry

Cite this

The role of β93 Cys in the inhibition of Hb S fiber formation. / Knee, Kelly M.; Roden, Catherine K.; Flory, Mark; Mukerji, Ishita.

In: Biophysical Chemistry, Vol. 127, No. 3, 01.05.2007, p. 181-193.

Research output: Contribution to journalArticle

Knee, Kelly M. ; Roden, Catherine K. ; Flory, Mark ; Mukerji, Ishita. / The role of β93 Cys in the inhibition of Hb S fiber formation. In: Biophysical Chemistry. 2007 ; Vol. 127, No. 3. pp. 181-193.
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AB - Recent studies have suggested that nitric oxide (NO) binding to hemoglobin (Hb) may lead to the inhibition of sickle cell fiber formation and the dissolution of sickle cell fibers. NO can react with Hb in at least 3 ways: 1) formation of Hb(II)NO, 2) formation of methemoglobin, and 3) formation of S-nitrosohemoglobin, through nitrosylation of the β93 Cys residue. In this study, the role of β93 Cys in the mechanism of sickle cell fiber inhibition is investigated through chemical modification with N-ethylmaleimide. UV resonance Raman, FT-IR and electrospray ionization mass spectroscopic methods in conjunction with equilibrium solubility and kinetic studies are used to characterize the effect of β93 Cys modification on Hb S fiber formation. Both FT-IR spectroscopy and electrospray mass spectrometry results demonstrate that modification can occur at both the β93 and α104 Cys residues under relatively mild reaction conditions. Equilibrium solubility measurements reveal that singly-modified Hb at the β93 position leads to increased amounts of fiber formation relative to unmodified or doubly-modified Hb S. Kinetic studies confirm that modification of only the β93 residue leads to a faster onset of polymerization. UV resonance Raman results indicate that modification of the α104 residue in addition to the β93 residue significantly perturbs the α1β2 interface, while modification of only β93 does not. These results in conjunction with the equilibrium solubility and kinetic measurements are suggestive that modification of the α104 Cys residue and not the β93 Cys residue leads to T-state destabilization and inhibition of fiber formation. These findings have implications for understanding the mechanism of NO binding to Hb and NO inhibition of Hb S fiber formation.

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