Lidstrom:Reducing Agents

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Why are reducing agents used?

  • Two cystines can either be paired in disulfide bonds (−S−S−) or can be reduced as two sulfhydryl groups (−SH).
    • If a cystine is catalytic, it needs to be in the reduced form.
  • Disulfide bonds in proteins are subject to cleavage by mild reducing agents, by a few oxidizing agents, or by nucleophilic displacement (citation)

Why are reducing agents a problem?

  • Recombinant proteins with highly reactive thiol groups can form disulfide adducts with reducing agents commonly used in protein purification, such as beta-mercaptoethanol and dithiothreitol. These adducts can interfere with protein-protein or protein-ligand interactions. (citation)

Why are reducing agents a problem?

  • Recombinant proteins with highly reactive thiol groups can form disulfide adducts with reducing agents commonly used in protein purification, such as beta-mercaptoethanol and dithiothreitol. These adducts can interfere with protein-protein or protein-ligand interactions.

What reducing agents are commonly used?

  • DTT
    • the most popular reducing agent for proteins (citation)
  • b-ME (β-mercaptoethanol)
    • foul odor, liquid
    • widely used in prior to 1970 before being replacing by DTT (citation)
  • TCEP (tris(2-carboxyl)phosphine)
    • more expensive
    • lacks odor (citation)
    • stronger reducing capacity and decreased probability of oxidation in air (citation)

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