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.

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$.$

Q$2$ 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

band in the protein