Hello. If you mean peroxidases (POX; EC 1.11.1.7), you can use the method based on nitrophenol generation from PNPP (p-nitrophenylphosphate) at 460 nm. In some cases also guaiacol is used (e.g. http://www.docstoc.com/docs/59527287/Assay-Procedure---Peroxidase).
I've been using the first one and I am glad with the results.
If you study green tissue, I recommend ascorbate peroxidase (APX; EC 1.11.1.11), it's a valuable method in some comparative studies.
If you want more details, please look into my papers I've uploaded on my profile, or I'll give them later - now I am on the trip.
Maybe the others also will help and discuss some pro and contras of using the methods :).
if you are assaying GPX activity then please use this method.
Assay and activity staining of guaiacol peroxidase (GPX, EC: 1.11.1.7)
Peroxidase activity was measured in a reaction mixture (3 ml) containing 100mM Na2HPO4 buffer pH 6.8 (1.5ml), 12mM H2O2 (0.5ml), 3mM guaiacol (0.5ml) and 0.5ml enzyme extract according to the method of Kar and Fierabend (1984).The increase in absorbance due to formation of tetra guaiacol was recorded at 470 nm. Peroxidase activity was calculated by using the extinction co efficient of 26.6M-1cm-1 for H2O2 at 470 nm and was expressed as nKat/ mg of protein.
10 % gel was stained by the procedure of Ulmer et al. (1971). Gels were immersed in 0.018M guaiacol for 30 mins at room temp. Rinsed twice in deionized water then immersed in 0.015% H2O2 in 1% glacial acetic acid. Band developed in about 10 min.
Hi,
Which oxydase?
If measuring polyphenoloxidase actually the prefered assay is polarographic (i.e. to follow disappearenace of the second substrate O2); the phenolic substrates are usually catechol, 4-methylcatechol (catecholase activity) and p-cresol (cresolase activity). Colorimetric assays also exist; the simplest is to measure the OD at 400 nm, but it reflects reaction products from the quinons and thus is far from the "true" actiivty (as measured directly by polarography). A better alternative is to use a molecule that reacts with the quinones to form coloured compounds. A popular method is Espin et al., J. food Sci 61 (1996) 1177-1181.
For lipoxygenase, increase in absorbance at 234 nm corresponding to generation of conjugated double bonds in linoleic acid is also a good colorimetric assay. Surrey, Plant Physiol 39 (1964) 65-70.
We need to know the specific type of oxidase you are referring to, as there are many different oxidases which may be determined by colorimetric method.
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