Hi, see this recent methods paper:

http://www.ncbi.nlm.nih.gov/pubmed/22656066

In the original paper (1974) when pyrogallol was first reported as a superoxide donor, it is said that catalase and H2O2 did not affect the pyrogallol autoxidation process. 

http://www.ncbi.nlm.nih.gov/pubmed/4215654

The modified method in the first link claims that the method works with a broad range of antioxidants. In your case, it is SOD inhibitors to be tested, and perhaps there are no such works (on cyanide) around. 

This paper below used azide:

http://www.ncbi.nlm.nih.gov/pubmed/7691133

Thank you Nikola for answer my question. In the case of H2O2 exist an interference with the metthod ? In need to evaluate the Cu-Zn activity to, wath can I do for this.

I have not done the inhibition by CN or H2O2 ib solution (liquid). And, I don't like to use the pyrogallol method to assay SODs. In my opinion, the best assay for determining the Specific activities of the different SODs is in PAGE activity stain - using NBT/Riboflavin and light to generate superoxide radicals that will reduce NBT fo formazan. The presence of an SOD will give an achromatic band. By soaking the native PAGE gel in the staining solution containing either CN- or H2O2 you can visually see which band is inhibited. For quantification, you need to load a known amount of cell extracts that has about 2-3 SOD units (maximum) in order to stay in the linear dynamic range  of the achromatic bands. If you scan the control untreated gel and integrate the areas under the peak for each isomer you can determine the contribution of each to the total area and hence the activity in each band. Now you can do the same for the cyanide and the H2O2 treated gels and determine the percent inhibition for each isomer. For the NBT assay in Gels, see [ Beauchamp C and Fridovich I (1971) Anal. Biochem. 44:276-287].

Hi Hosni M Hassan .

Than you for your long and very interesting answer. we work with vegetables extracts and in this field there are not papers about SOD quantification of isoform in solution, yes there are works using PAGE activity stain but in our countri is more dificult make a gel tha measure yhe activity in a spectrophotemeter. For this reasons we are researching in this theme.

Superoxide dismutase was so easy to be dtermined by Murkland....

u should prepare ur buffer (Tris-HCl 0.05 M containing 1 mM DTPA pH 8.5, this can be prepared by calculating the wt of Tris and DTPA to have their concentration but don't complete the volume to the end i.e. leave a small water amount before adjusting the total volume... now adjust pH to 8.5 by HCl then add the small water amount remained to reach the total volume)

pyrogallol prepared 20 mM in 10 mM HCl

depending on ur tissue SOD level the volume of buffer, sample and pyrogallol added to the reaction mixture can be determined... for example, u can use 1 ml buffer and 10 or 20 ul of ur homogenate then add 10 ul of pyrogallol... at the same time u put pyrogallol and mix it well with the reaction mixture, adjust ur stopwatch and measure the absorbance at 420 nm at timed intervals (may be 1, 2 and 3 min readings or 1.5, 2.5 and 3.5 min readings... u can take both and calculate the activity which may give u the best results)

for calculating the activity after that... u should read blank first (buffer + water or ur homogenizing buffer + pyrogallol) that should read higher than ur samples...

then calculate dA/dt = ((reading at last interval - reading at second interval)+(reading at second interval - reading at first interval))/2 min.... the duration u take readings in.....

then calculate the inhibition percent = (dA/dt of blank - dA/dt of sample) / dA/dt of blank * 100

u should do the same for a series of sod standard solutions prepared in deionized water.... and test ur concentrations to know the concentration range u can take it for measuring....

if u find that dA/dt of samples are so small or nearly zero, u should dilute ur samples...

and if u find that dA/dt of ur samples are higher than that of blank, so u should increase ur sample volume or contration in the reaction mixture.... ur accepted range of inhibtion percent should be between 20% - 60%..... if it is higher, dilute ur samples...

if smaller, increase ur sample volume...

then construct a calibration curve, and get the sod concentration at which 50% inhibtion is achieved...

this concentration can be considered as 1 U

from this u can get the activity in units.... then divide it by the protein concentration in ur homogenate, so u can get the specific activity....

i hope i could give u a detailed summary of the procedure....

u just need to standardize ur assay depending on ur tissue level of sod....

N.B. if u calculate the dilution factor of pyrogallol in the reaction mixture u will find its final concentration is 0.2 mM

don't hesitate to ask about anything

I think the above answers were useful