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Is it possible to replace 3-mercaptopropionic acid with 2-mercaptoethanol to solubilize avian eggshell membrane (ESM) to soluble eggshell membrane protein (SEP)?

Most literature suggested the use of mercaptopropionic acid but I only have mercaptoethanol in the lab. This is because both of them works as reducing agent to cleave the disulphide bonds to solubilize the protein.

Will it works without denaturing the protein?

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    $\begingroup$ If you are breaking the disulfide bonds, you are denaturing the protein. $\endgroup$
    – matt_black
    Mar 10, 2023 at 11:19

1 Answer 1

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Mercaptoethanol is the reagent used as denaturing agent for protein thus you can use it for this purpose. The mechanism for denaturing proteins by mercaptoethanol is to disrupt the disulfide bonds found in the protein. Therefore, using it will denature the protein in concern (I think same result you would achieve by using 3-mercaptopropionic acid).

Any mercapto-group $(\ce{R-SH})$ would disrupt disulfide bond $(\ce{R-S-S-R'})$. For example, cysteine and glutathione (GSH) have been used in vitro to denature disulfide bonds (Ref.1). In a typical procedure:

The compounds were incubated at $\pu{10 mM}$ with $0.03$ and $\pu{0.2 mM}$ cysteine or $\pu{4 mM}$ GSH, in $\pu{100 mM}$ Tris buffer, $\mathrm{pH} \ 7.0$, containing 5% methanol at $\pu{37 ^\circ C}$. Aliquots were taken at $0, 1, 4,$ and $\pu{24 hours}$, and the samples were analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS).

Also read Ref. 2 & 3 for more details.

References:

  1. Donglu Zhang, Aimee Fourie-O’Donohue, Peter S. Dragovich, Thomas H. Pillow, Jack D. Sadowsky, Katherine R. Kozak, Robert T. Cass, Liling Liu, Yuzhong Deng, Yichin Liu, Cornelis E.C.A. Hop, and S. Cyrus Khojasteh, "Catalytic Cleavage of Disulfide Bonds in Small Molecules and Linkers of Antibody–Drug Conjugates," Drug Metabolism and Disposition 2019, 47(10), 1156-1163 (DOI: https://doi.org/10.1124/dmd.118.086132).
  2. Oliver Smithies, "Disulfide-Bond Cleavage and Formation in Proteins," Science 1965, 150(3703), 1595-1598 (DOI: 10.1126/science.150.3703.159).
  3. Navin Kumar D. Kella and John E. Kinsella, "Disulfide-Bond Cleavage and Formation in Proteins," Journal of Biochemical and Biophysical Methods 1985, 11(4-5), 251-263 (DOI: https://doi.org/10.1016/0165-022X(85)90007-7).
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