HRP and TMB - the chemistry (Feb/16/2009 )
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?
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.
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
Sorry for the typo its Diimine and not diamine
Diamine is parent molecule and diimine is the stable oxidatn product
TMB-reagent without perhydrol
Phosphate-Citrate Buffer 0.1 M at pH 4.6 ± 0.2 plus perborate : mix 24 ml of 0.2 M dibasic sodium phosphate (dissolve 1.78 g Na2HPO4-2H2O or 3.58 g of Na2HPO4•12H2O in dH2O e fill up to 50 ml) with 26 ml 0.1 M citric acid (2.10 g citric acid monohydrate in dH2O and make to 100 ml with dH2O), check pH and dissolve 0.02 g sodium perborate. Store in polyethylene bottle at 4 °C stable 1 year.
TMB substrate 100 mM : dissolve 240 mg 3,3’,5,5’–tetramethylbenzidine (m.w. 240) in 10 ml of DMSO. Store in opaque polyethylene bottle at 4 °C stable 1 year.
Work TMB chromogen : mix 25 µl of TMB substrate to 1.0 ml of phosphate-citrate buffer plus perborate, stable for several hours at room temperature or for few days at 4 °C.