At pH 3.5 to pH 4.5 which is the better and efficient reducing agent to break all 4 disulfide bonds in my peroxidase enzyme? Also which reducing agent and concentration is compatible with far-UV CD? MS results show that atleast 20 mM Dtt is needed to break all disulfide bonds. Is this concentration compatible with cd scan from 200 nm to250 nm and melting curve at 222 nm? I think maybe 20 mM TCEP is better than Dtt for far-uv cd? Is TCep supposed to be better under low pH (3.5 - 4.5) ? Any ideas are welcome.
Dear Khawar,
Thiol groups reduce your compounds through a mechanism that involves two consecutive nucleophilic attacks of thiolate to a disulfide bridge. The lower the concentration of thiolate (ionized form) the slower the reaction. For that reason, DTT and 2-mercaptoethanol are clearly ineffective at acidic pH values (pKa above 7.5 in aqueous solution). For the pH range you want, you need to use TCEP which is a phosphine derivative and has a mechanism much less affected by pH. TCEP has been demostrated to be effective a pH4.5 and 5, while DTT is almost uneffective. Check at this reference:
Getz EB , Xiao M , Chakrabarty T , Cooke R , & Selvin PR (1999) A Comparison between the Sulfhydryl Reductants Tris(2-carboxyethyl)phosphine and Dithiothreitol for Use in Protein Biochemistry. Analytical Biochemistry 273(1):73 - 80.
Best wishes,
Rogelio
You must use TCEP. DTT loses its reducing power at a ph lower then 7.0. Also DTT breaks down rapidly while TCEP is stable.
I would go for TCEP. But be aware that TCEP is at least 10x more expensive than DTT, and making liters of buffers with TCEP, can become an expensive affair.
TCEP is more suitable at described conditions and much more friendly than DTT as well. TCEP efficiency also goes down at lower pH but definitely better than DTT. Moreover TCEP works much faster as well.
Why not beta-mercaptoethanol? Cheaper and probably you do not need 20 mM, as it seems more effective thatn DTT. Otherwise, I agree with other concerning TCEP and carboxyamidation ---- with iodoacetamide? But that approach is irreversible.
Dear Khawar,
Thiol groups reduce your compounds through a mechanism that involves two consecutive nucleophilic attacks of thiolate to a disulfide bridge. The lower the concentration of thiolate (ionized form) the slower the reaction. For that reason, DTT and 2-mercaptoethanol are clearly ineffective at acidic pH values (pKa above 7.5 in aqueous solution). For the pH range you want, you need to use TCEP which is a phosphine derivative and has a mechanism much less affected by pH. TCEP has been demostrated to be effective a pH4.5 and 5, while DTT is almost uneffective. Check at this reference:
Getz EB , Xiao M , Chakrabarty T , Cooke R , & Selvin PR (1999) A Comparison between the Sulfhydryl Reductants Tris(2-carboxyethyl)phosphine and Dithiothreitol for Use in Protein Biochemistry. Analytical Biochemistry 273(1):73 - 80.
Best wishes,
Rogelio
It is a matter of basic chemistry! if you write down the redox reaction to form disulfide connections you will see that at acidic pH the protons are involved and will not favour the reaction to keep cystin bridges open.
Thanks for all the wonderful suggestions. My enzyme is a metalloprotein. Is it possible for TCEP to reduce metals such as Mn+++ and Fe+++ to Mn++ and Fe++ in addition to reducing disulfide bridges??
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