Determination of —SH Groups in Proteins

Benesch, Ruth E.

doi:10.1002/9780470110270.ch2

Cited by 161 publications

(12 citation statements)

References 74 publications

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“…Previously, modifications at Hb Cys residues other than β93 were only observed at high concentrations of modifying reagent [14,15,31]. Benesch and co-workers [31] observed that titration with an 100-fold molar excess of PMB results in the observation of 7.5 reactive -SH groups per mole of Hb, while lower PMB concentrations yield a value of 2.4 reactive -SH groups per mole of Hb. More recently, Ferranti and coworkers [14] demonstrated by ESI-MS that a 100-fold excess of NO relative to Hb leads to S-nitrosothiol formation at the α104 and β112 Cys residues.…”

Section: The α104 Cys Residue Is Modified By Nemmentioning

confidence: 98%

“…The peak intensities of individual species were compared, and these analyses revealed that 33± 11% of the total α chain intensity of NEM-H Hb corresponds to modified α chain, while only 12± 7% of the total α chain intensity of NEM-L Hb can be assigned to modified α chain ( Table 2). Modification of the α-chain was considered to occur at the α104 Cys residue only because of the selective reactivity of the NEM group for Cys residues under the reaction conditions used [29][30][31][32].…”

Section: Electrospray Ionization Mass Spectrometrymentioning

confidence: 99%

“…A variety of different methods, including FT-IR spectroscopy, ESI-MS and tandem MS, indicate NEM modification occurs at both β93 and α104 Cys residues and that the α104 residue is moderately reactive under relatively standard conditions [37]. Previously, modifications at Hb Cys residues other than β93 were only observed at high concentrations of modifying reagent [14,15,31]. Benesch and co-workers [31] observed that titration with an 100-fold molar excess of PMB results in the observation of 7.5 reactive -SH groups per mole of Hb, while lower PMB concentrations yield a value of 2.4 reactive -SH groups per mole of Hb.…”

Section: The α104 Cys Residue Is Modified By Nemmentioning

confidence: 99%

“…The choice of NEM as the modifying agent stems from its specific reactivity with Cys sulfhydryl groups under conditions of neutral pH [29]. In addition, previous work had indicated that only the β93 Cys residues were reactive out of the six Cys residues present in an Hb tetramer [30][31][32]. Chemical modification of β93 Cys simulates SNO formation at β93 without the possibility of other reactions, such as formation of HbNO or methemoglobin.…”

Section: Introductionmentioning

confidence: 99%

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

Knee

Roden

Flory

et al. 2007

Biophysical Chemistry

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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 singlymodified 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 α 1 β 2 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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Section: The α104 Cys Residue Is Modified By Nemmentioning

confidence: 98%

Section: Electrospray Ionization Mass Spectrometrymentioning

confidence: 99%

Section: The α104 Cys Residue Is Modified By Nemmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

Knee

Roden

Flory

et al. 2007

Biophysical Chemistry

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show abstract

“…Hughes et al, 1950;Perkins, 1952Perkins, , 1953Perkins, , 1961Hughes. 1957;Passow et al, 1961;Benesch & Benesch, 1962). The decreased uptake in muscular tissue which was observed when higher doses were given may be due to an initial saturation of available sites for the compound and thus a lack of free groups with affinity for methyl mercury.…”

Section: Level Of Dosagementioning

confidence: 99%