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1. The sidechains of internal cysteine amino acids undergo oxidation to produce disulfide crosslinks that stabilize the protein structure. Solvent-exposed cysteine sidechains can remain in their reduced thiol form and serve as reactive nucleophiles (recall for instance Acyl Carrier Proteins involved in fatty acid biosynthesis or various oxidoreductase enzymes). The reactive solvent-exposed thiol groups in proteins can be detected using Ellman's reagent. The thiol reacts with the disulfide Ellman's reagent to form a new mixed disulfide and a 2-nitro- 5-thiobenzoate (TNB) byproduct. Upon addition of base, TNB is deprotonated to form a dianion, which absorbs light in the visible region and can be detected using UV-Vis spectroscopy. Therefore the production of TNB can be correlated with surface-exposed SH. Ellman's Reagent CO₂H -NO2 internal disulfide crosslink O₂N- S-S HO₂C SH. solvent- exposed thiol UV Absorbance at 412 nm O₂N- 020 TNB2- alkaline pH O₂N- -SH HO₂C 2-nitro-5-thiobenzoate (TNB) -NO₂ mixed disulfide CO₂H a. Provide a detailed arrow-pushing mechanism for the reaction of a thiol (you can use R-SH) with Ellman's reagent to produce a mixed disulfide and TNB. Ellman's Reagent CO₂H -NO₂ ,, ༣ R-SH O₂N- HO₂C R-S CO₂H -NO2 O₂N- -SH HO₂C TNB Mixed disulfide

1. The sidechains of internal cysteine amino acids undergo oxidation to produce disulfide crosslinks that stabilize the protein structure. Solvent-exposed cysteine sidechains can remain in their reduced thiol form and serve as reactive nucleophiles (recall for instance Acyl Carrier Proteins involved in fatty acid biosynthesis or various oxidoreductase enzymes). The reactive solvent-exposed thiol groups in proteins can be detected using Ellman's reagent. The thiol reacts with the disulfide Ellman's reagent to form a new mixed disulfide and a 2-nitro- 5-thiobenzoate (TNB) byproduct. Upon addition of base, TNB is deprotonated to form a dianion, which absorbs light in the visible region and can be detected using UV-Vis spectroscopy. Therefore the production of TNB can be correlated with surface-exposed SH. Ellman's Reagent CO₂H -NO2 internal disulfide crosslink O₂N- S-S HO₂C SH. solvent- exposed thiol UV Absorbance at 412 nm O₂N- 020 TNB2- alkaline pH O₂N- -SH HO₂C 2-nitro-5-thiobenzoate (TNB) -NO₂ mixed disulfide CO₂H a. Provide a detailed arrow-pushing mechanism for the reaction of a thiol (you can use R-SH) with Ellman's reagent to produce a mixed disulfide and TNB. Ellman's Reagent CO₂H -NO₂ ,, ༣ R-SH O₂N- HO₂C R-S CO₂H -NO2 O₂N- -SH HO₂C TNB Mixed disulfide

Chemistry
Chemistry
10th Edition
ISBN:9781305957404
Author:Steven S. Zumdahl, Susan A. Zumdahl, Donald J. DeCoste
Publisher:Steven S. Zumdahl, Susan A. Zumdahl, Donald J. DeCoste
Chapter1: Chemical Foundations
Section: Chapter Questions
Problem 1RQ: Define and explain the differences between the following terms. a. law and theory b. theory and...
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1. The sidechains of internal cysteine amino acids undergo oxidation to produce disulfide crosslinks that stabilize the protein structure. Solvent-exposed cysteine sidechains can remain in their reduced thiol form and serve as reactive nucleophiles (recall for instance Acyl Carrier Proteins involved in fatty acid biosynthesis or various oxidoreductase enzymes). The reactive solvent-exposed thiol groups in proteins can be detected using Ellman's reagent. The thiol reacts with the disulfide Ellman's reagent to form a new mixed disulfide and a 2-nitro- 5-thiobenzoate (TNB) byproduct. Upon addition of base, TNB is deprotonated to form a dianion, which absorbs light in the visible region and can be detected using UV-Vis spectroscopy. Therefore the production of TNB can be correlated with surface-exposed SH. Ellman's Reagent CO₂H -NO2 internal disulfide crosslink O₂N- S-S HO₂C SH. solvent- exposed thiol UV Absorbance at 412 nm O₂N- 020 TNB2- alkaline pH O₂N- -SH HO₂C 2-nitro-5-thiobenzoate (TNB) -NO₂ mixed disulfide CO₂H a. Provide a detailed arrow-pushing mechanism for the reaction of a thiol (you can use R-SH) with Ellman's reagent to produce a mixed disulfide and TNB. Ellman's Reagent CO₂H -NO₂ ,, ༣ R-SH O₂N- HO₂C R-S CO₂H -NO2 O₂N- -SH HO₂C TNB Mixed disulfide
Transcribed Image Text:1. The sidechains of internal cysteine amino acids undergo oxidation to produce disulfide crosslinks that stabilize the protein structure. Solvent-exposed cysteine sidechains can remain in their reduced thiol form and serve as reactive nucleophiles (recall for instance Acyl Carrier Proteins involved in fatty acid biosynthesis or various oxidoreductase enzymes). The reactive solvent-exposed thiol groups in proteins can be detected using Ellman's reagent. The thiol reacts with the disulfide Ellman's reagent to form a new mixed disulfide and a 2-nitro- 5-thiobenzoate (TNB) byproduct. Upon addition of base, TNB is deprotonated to form a dianion, which absorbs light in the visible region and can be detected using UV-Vis spectroscopy. Therefore the production of TNB can be correlated with surface-exposed SH. Ellman's Reagent CO₂H -NO2 internal disulfide crosslink O₂N- S-S HO₂C SH. solvent- exposed thiol UV Absorbance at 412 nm O₂N- 020 TNB2- alkaline pH O₂N- -SH HO₂C 2-nitro-5-thiobenzoate (TNB) -NO₂ mixed disulfide CO₂H a. Provide a detailed arrow-pushing mechanism for the reaction of a thiol (you can use R-SH) with Ellman's reagent to produce a mixed disulfide and TNB. Ellman's Reagent CO₂H -NO₂ ,, ༣ R-SH O₂N- HO₂C R-S CO₂H -NO2 O₂N- -SH HO₂C TNB Mixed disulfide
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