Shreffler:Pervanadate

Overview

Pervanadate is the peroxidized form of vanadate -- an oxoanion of vanadium. It acts as an irreversible inhibitor of the family of protein tyrosine phosphatases (PTPs) with an IC50 of ~0.3 μM by oxidizing a cysteine at the enzymatic active site.

Materials

• H₂O
• HEPES, 20 mM
• Na₃VO₄
• Catalase
• Hydrogen Peroxide (H₂O₂), 30%.

Procedure

1. Take one tube of 100 mM sodium orthovanadate Na₃VO₄ (Must be white/colorless. If it shows yellow, means VO₄ forms 12-mer, cannot use.)
2. Prepare 20 mM HEPES adjust pH to 7.3
3. Take 30% H₂O₂ dilute 10 times with HEPES buffer, 5 μL 30% H₂O₂ + 45 μL HEPES buffer, mix gently.
4. Dilute 3% H₂O₂ 10 times further with HEPES buffer, 5 μL 3% H₂O₂ + 45 μL HEPES buffer, mix gently.
5. Add in 10 μL 100 mM Na₃VO₄, 940 μL H₂O. Mix by gently reversing the vial. This will make up a 1 mM pervanadate stock.
6. After 5 minutes, use the tip of pipet to scoop a small amount of catalase, mix in to the pervanadate stock. This will result in a burst of O₂ bubbles. Open the lid of the tube to release the air pressure.
7. The pervanadate is good for 2 hours.

Discussion

discuss this protocol

References

1. Huyer G, Liu S, Kelly J, Moffat J, Payette P, Kennedy B, Tsaprailis G, Gresser MJ, and Ramachandran C. Mechanism of inhibition of protein-tyrosine phosphatases by vanadate and pervanadate. J Biol Chem. 1997 Jan 10;272(2):843-51. DOI:10.1074/jbc.272.2.843 |

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