I performed the peroxidase activity assay from certain leaf extracts using pyrogallol as susbtrate. I have the absorbance values but am facing the problem in determining the enzyme activity, because I am unsure of the extinction coefficient for purpurogallin to be used for the calculation. Various research articles have stated various values for the same. (The most commonly cited article by Chance and Maehly (1955) is unavailable).
If the reaction is rapid and stoichiometric, and has a low Km for hydrogen peroxide, then you can determine the extinction coefficient from the total change in absorbance on adding small known amounts of peroxide. You can determine the concentration of hydrogen peroxide using an extinction coefficient of 43.6 /M-cm (not /mM-cm!) at 240 nm, from http://www.bio.net/bionet/mm/methods/1995-January/023533.html.
http://www.bio.net/bionet/mm/methods/1995-January/023533.html
The molar extinction coefficient of purpurogallin ε420nm is 2640 M-1.cm-1 in 50 mM pH 7.0 phosphate buffer, 4400 M-1.cm-1 in toluene, 4320 M-1.cm-1 in acetonitrile, and 4260 M-1.cm-1 in ethylacetate, respectively.
As previously mentioned, the molar extinction coefficient of purpurogallin ε420nm is 2640 M-1.cm-1 in 50 mM pH 7.0 phosphate buffer, 4400 M-1.cm-1 in toluene, 4320 M-1.cm-1 in acetonitrile, and 4260 M-1.cm-1 in ethylacetate, respectively.
If you are using another buffer, you should get your own calibration curve to get the molar extinction coefficient under the conditions you are working. Getting the calibration curve is simple, prepare purpurogallin in the buffer you are using and check the absorbance at diferent wavelengths. Then plot the absorbance against the wavelength. The slope you get is de molar extinction coefficient.
@Deepti Ma'am, @Rosa Ma'am Thank you for your help !
@Edward Sir thank you for providing the extinction coeff. of H2O2. I will be needing that for the Catalase assay.
- Badges
- Science method