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Question 2. While internal cysteine amino acid sidechains in proteins undergo oxidation to produce disulfide crosslinks that stabilize the protein structure after folding, 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 thiol groups in proteins can be detected using Ellman's test, wherein the protein is reacted with a 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. Ellman's Reagent CO₂H -NO₂ internal disulfide crosslink O₂N- HO₂C SH solvent- exposed thiol UV Absorbance at 412 nm O₂N- -S 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 R-SH + O₂N- HO₂C CO₂H -NO2 R-S Mixed disulfide CO₂H + -NO₂ O₂N- -SH HO₂C TNB Q2 Cont'd: b) Provide a reasonable explanation for why the TNB byproduct does not react further to undergo a similar type of disulfide exchange with the internal disulfide bond in the protein. -S + -NO₂ mixed disulfide CO₂H O₂N- -SH X “མ་ HO₂C TNB CO₂H NO2 SH S -NO2 CO₂H

Question 2. While internal cysteine amino acid sidechains in proteins undergo oxidation to produce disulfide crosslinks that stabilize the protein structure after folding, 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 thiol groups in proteins can be detected using Ellman's test, wherein the protein is reacted with a 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. Ellman's Reagent CO₂H -NO₂ internal disulfide crosslink O₂N- HO₂C SH solvent- exposed thiol UV Absorbance at 412 nm O₂N- -S 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 R-SH + O₂N- HO₂C CO₂H -NO2 R-S Mixed disulfide CO₂H + -NO₂ O₂N- -SH HO₂C TNB Q2 Cont'd: b) Provide a reasonable explanation for why the TNB byproduct does not react further to undergo a similar type of disulfide exchange with the internal disulfide bond in the protein. -S + -NO₂ mixed disulfide CO₂H O₂N- -SH X “མ་ HO₂C TNB CO₂H NO2 SH S -NO2 CO₂H

Organic Chemistry: A Guided Inquiry
Organic Chemistry: A Guided Inquiry
2nd Edition
ISBN:9780618974122
Author:Andrei Straumanis
Publisher:Andrei Straumanis
Chapter27: Amines
Section: Chapter Questions
Problem 5E
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Question 2. While internal cysteine amino acid sidechains in proteins undergo oxidation to produce disulfide crosslinks that stabilize the protein structure after folding, 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 thiol groups in proteins can be detected using Ellman's test, wherein the protein is reacted with a 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. Ellman's Reagent CO₂H -NO₂ internal disulfide crosslink O₂N- HO₂C SH solvent- exposed thiol UV Absorbance at 412 nm O₂N- -S 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 R-SH + O₂N- HO₂C CO₂H -NO2 R-S Mixed disulfide CO₂H + -NO₂ O₂N- -SH HO₂C TNB
Transcribed Image Text:Question 2. While internal cysteine amino acid sidechains in proteins undergo oxidation to produce disulfide crosslinks that stabilize the protein structure after folding, 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 thiol groups in proteins can be detected using Ellman's test, wherein the protein is reacted with a 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. Ellman's Reagent CO₂H -NO₂ internal disulfide crosslink O₂N- HO₂C SH solvent- exposed thiol UV Absorbance at 412 nm O₂N- -S 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 R-SH + O₂N- HO₂C CO₂H -NO2 R-S Mixed disulfide CO₂H + -NO₂ O₂N- -SH HO₂C TNB
Q2 Cont'd: b) Provide a reasonable explanation for why the TNB byproduct does not react further to undergo a similar type of disulfide exchange with the internal disulfide bond in the protein. -S + -NO₂ mixed disulfide CO₂H O₂N- -SH X “མ་ HO₂C TNB CO₂H NO2 SH S -NO2 CO₂H
Transcribed Image Text:Q2 Cont'd: b) Provide a reasonable explanation for why the TNB byproduct does not react further to undergo a similar type of disulfide exchange with the internal disulfide bond in the protein. -S + -NO₂ mixed disulfide CO₂H O₂N- -SH X “མ་ HO₂C TNB CO₂H NO2 SH S -NO2 CO₂H
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