Read the attached files.

Beast regards

Esam Mansour

Article A Simple Colorimetric Method for the Measurement of Hydrogen...

Article A simple colorimetric method for determination of hydrogen p...

Article A colorimetric method of analysis for trace amounts of hydro...

Guaiacol peroxidase (GPOX; EC 1.11.1.7) activity was

measured according to Fielding and Hall (1978). This

method is based on monitoring the GPOX scavenging

activity by using guaiacol as a hydrogen donor. The

reaction mixture contained 10 mM H2O2 in 50 mM

phosphate buffer (pH 7), 9 mM guaiacol and the

enzyme extract in a total volume of 500 ml. GPOX

activity was estimated by the increase in the absorbance

of tetra-guaiacol at 470 nm and was expressed as

micromole of guaiacol oxidized per min at 258C.

smiri et al. 2013. Journal of Plant Interactions, 2013 Vol. 8, No. 3, 255262,

Peroxidases catalyzes the dehydrogenation of compounds such as o-dianisidine, guaiacol etc.

The below mentioned method is using o-dianisidine-

Reaction mixture comprises of 0.003% hydrogen peroxide in 0.1 M phosphate buffer, pH 6. 0, 0.01% o-dianisidine dye (dissolve in methanol). An appropriate amount of crude enzyme extract is added (often one needs to dilute the same) and the increase in absorbance is recorded at 460nm.

In addition to conventional procedure for the determination of peroxidase activity, one can use some natural substrates widely present in the leaf tissue, such as phenolics. Please read the attached file. The described procedure is applicable to the leaf tissue also.

1. pipette out 3 mL of buffer solution, 0.05 mL guaiacol solution, 0.1 mL enzyme extract and 0.03 mL H2O2 solution in a cuvette.

2. Mix well. Place the cuvette in the spectrophotometer.

3. Wait until the absorbance has increased by 0.05. Start a stop watch and note time required in minutes (change in t) to increase the absorbance by 0.1. 

for calculation and other information refer to Biochemical Methods

book By S Sadasivam. 

 Can I use the simple enzyme extract made by phosphate buffer and EDTA.Then Add TCA directly into the supernatant.. allowed in the dark for 1hr and calculate absorbtion??