@SHACKIRA AM  

I tried this i used the same sample and tested three times each time i got a different result with the high difference, i prepared. i used distilled water to prepare the H2O2 solution and Ascorbate might i made mistake there i will try it again. thank you.

Today i again tried method elaborated by Nakano and Asada, i used same sample three times and every time i got the reading different from previous. can anybody suggest men another method for APX.

A few things you can try:

• Add ascorbic acid to the homogenization/extraction buffer to avoid loss of activity;
• Check if your samples have high ascorbate oxidase activity, which interferes in the assay. This is done by monitoring ascorbate at 290 nm in an assay containing assay buffer, ascorbic acid and sample;
• Make a control experiment containing assay buffer, hydrogen peroxide and ascorbic acid to account for the non-enzymatic oxidation of ascorbate;
• Check the concentration of your hydrogen peroxide solution at 240 nm;
• Check the concentration of your ascorbic acid solution at 265 or 290 nm.

Take a look at: Asada K (1984) Chloroplasts: Formation of active oxygen and its scavenging. Meth Enzymol 105:422–429. doi: 10.1016/S0076-6879(84)05059-X

Dear @Daniel Carneiro Moreira how much concentration of hydrogen peroxide solution at 240 nm should be?

And how much concentration of  ascorbic acid solution at 265 or 290 nm?

Looking for your experties. thanks you 

The molar attenuation coefficient for hydrogen peroxide at 240 nm is 0.04 mM-1 cm-1. Thus, a 1 mM solution (in a 1 cm path length cuvette) has an absorbance of 0.04.

In the case of ascorbic acid the coefficient at 290 nm is 2.8 mM-1 cm-1. Thus, a 0.1 mM solution (in a 1 cm path length cuvette) has an absorbance of 0.28.

Thank you so much, dear, @ Daniel Carneiro Moreira. Your help really appreciated 

Dear Fatemeh Gholizadeh

Please find below the details for measuring Ascorbate Peroxidase (APX) that I have used recently following the method of Nakano and Asada (1981).

A. Extraction:

o The leaf tissues were collected from different treated sets after 24 h incubation and homogenized with liquid nitrogen.

o 0.5 mg of powdered sample was extracted with 2 ml of Sodium phosphate buffer (ph 7.0, 0.1M) containing 0.1 % polyvinylpyrrolidone (PVP).

o All the extraction procedures were conducted at 4 °C.

o The homogenate was centrifuged at 11,0009g for 20 min at 4 C.

o The supernatants were used as the crude enzyme source for the enzymatic assays.

o Then it was transferred to a 2 ml Eppendorf tube and stored at -80 C for further use,

B. Dosage :

o The reaction mixture contained 50 mM potassium phosphate (pH 7.0), 0.2 mM EDTA, 0.5 mM ascorbic acid, 2 % H2O2, and 0.1 mL enzyme extract in a final volume of 3 mL.

o The decrease in absorbance at 290 nm for 1 min was recorded and the amount of ascorbate oxidized was calculated using extinction coefficient (e = 2.8 mM-1 APX was defined as 1 mmol mL-1 per min at 25 C, cm-1). Enzyme activity was expressed as umol min-1 g-1 FW.

I attached one file explaining protocols and how to calculate enzymes including APX.

Good luck,

Noureddine