Dear Amir,

Homogenize samples with 0.5% TCA in ice cold conditions and centrifuge homogenate at 15000 xg at 4°C. Store supernatant at 4°C till you use it for determination of H2O2. However, for peroxidase assay you need to carefully neutralize or adjust pH to that optimal for peroxidase activity. You may even use 0.1% TCA for homogenization.

Dear  P. Pardha-Saradhi,

I did not get your answer. Can you plesae re-send it againThanks,Amir

Dear  P. Pardha-Saradhi,

Thanks for your reply. Do you think we can use Na3PO4 instead of 0.5% TCA ?

Dear Amir,

I am sorry. I could not have access to network from last four days. We prefer extracting H2O2 in 0.5% trichloroacetic acid (TCA) at 4°C. We have never used NA3PO4, so I cannot give any definite answer. However, I believe that Na3PO4 would be alkaline and under alkaline conditions H2O2 may not be stable for long time (alkaline conditions promote formation of perhydroxylanion from H2O2). Yes, you may use Na3PO4 for neutralizing 0.5% 0r 0.1% extracted H2O2.

Hello,  this method included the extraction and determination of H2O2. I hope it will be helped you

Hydrogen peroxide (H2O2) concentration was determined according to Loreto and Velikova (2001). Leaf samples of 0.5 g were homogenized in 3 mL of 1% (w/v) tri-chloroacetic acid (TCA). The homogenate was centrifuged at 10,000 rpm and 4°C for 10 min. Subsequently, 0.75 mL of the supernatant was added to 0.75 mL of 10 mM K-phosphate buffer (pH 7.0) and 1.5 mL of 1M KI. H2O2 concentration of the supernatant was evaluated by comparing its absorbance at 390 nm to a standard calibration curve. The concentration of H2O2 was calculated from a standard curve plotted in the range from 100 to 1000 μmol mL-1. H2O2 concentration was expressed as μmol g-1 DW.

Ref.

Loreto F, Velikova V (2001) Isoprene produced by leaves protects the photosynthetic apparatus against ozone damage, quenches ozone products, and reduces lipid peroxidation of cellular membranes. Plant Physiol 127: 1781-1787

Hi Mohamed ,

Thanks for your message.I will try TCA.