4 replies to this topic

[jayesh]

Can anyone tell me what is the chemistry reaction between horse raddish peroxidase HRP and tetramethylbenzemidine TMB?
Why do we need hydorgen peroxidase and finally why do we stop the reaction with sulphuric acid?

jayesh

[genehunter]

http://www.nuncbrand.com/NAG/DP0011.htm#3%...nzidine%20(TMB)


HRP speeds up an electron transfer reaction. TMB is the donor and gets oxidized, and H2O2 is the recipient of the electron and gets reduced to O2 and H2O. so, H2O2 is one of the substrate. H2SO4 kills the enzyme as proteins dont survive under an extreme pH.

[LOW]

If the acid just to denature the HRP, then why the blue colour of oxidized TMB changes yellow?

[garry]

The acidic pH stabilizes the diamine form of TMB..so we have to stop it using H2SO4 or Phosphoric acid or HCl
The kit I'm using has Phosphoric acid..
Basically TMb is oxidised over two step 2e- oxidtn
and the stable form has this yellow color read at 450 I tried working out the resonance structures but not so sure..if someone's interested write to me I'll send u my structures for correction in ISIS draw files

[garry]

Sorry for the typo its Diimine and not diamine
Diamine is parent molecule and diimine is the stable oxidatn product

Edited by garry, 23 February 2010 - 08:03 AM.