I'm trying to quantify H2O2 in leaf extracts, according to Wang et al, 2012 and Patterson et al 1984. My problem is that I can't do the standard curve. I'm doing it with H2O2 30%, dilute to have different concentrations and have a good curve in 240nm. However, when I had titanium chloride and ammonia solution, and read it at 410nm, I don't have a curve, all the standard concentrations are more or less identical. I've already tried different approaches and I can't solve the problem.
Can anyone help me with this?
There is a spectrophotometric assay based on the ferrous oxidation in the presence of xylenol orange. The following paper can help you.
http://link.springer.com/content/pdf/10.1007%2Fs10059-010-0082-3.pdf
Thank you both for your answers, and sorry for my delay...
Ivan, I will look for that papper know, do you follow this method?
Jan, this AmphexRed/HRP method is some kind of kit? Do you have any paper were I can find the description of it?
Thank you!
Hello Monya, I used this method and got success to detect the H2O2 in the skin of fruits. It represents a simple way to detect H2O2... But I agree with Jan that the use of kits like this (Amplex) may be a good alternative too.
This article can be of some use for you: http://www.sciencedirect.com/science/article/pii/S0168945299001971
Hello Abdul,
Thank you for the article, in fact it was very useful, because I could quantify the peroxide, trough its methodologies!
Thanks!
Jan and Ivan,
Thank you for your contributions, they were very helpful!
Best regards!
I homogenize rice leaves into powder with liquid nitrogen then extract H2O2 with 5% w/v metaphosphoric acid solution (1 g leaf fresh weight per 6 mL extractant), centrifuge (22,000 xg, 4 oC, 20 minutes) and adjust the pH to 6.5 with 1M tricine in 6M NaOH. For the standards, I prepare different concentrations of H2O2 (0, 2, 4, 6, and 8 mM) then use same volume (100 uL) of standard and sample extract to assay H2O2 in a total of 1 mL together with the other assay components, done indirectly by measuring optical density (OD) at wavelength 340 nm. The amount of NADH available before (reflected in optical density, OD1)and after (reflected in optical density, OD2) the addition of NADH peroxidase to effect the stoichiometry of the reaction between H2O2 and 2NADH to form 2H2O and 2NAD+. The amount of NADH consumed in the reaction is the difference before and after the reaction (OD1-OD2). The standard curve is constructed by plotting amount of H2O2 (nmoles) present in the aliquot of standard used in the assay (x-axis) and OD1-OD2 (y-axis). Some relevant references are: Ella ES, Kawano N and Ito O. 2003. Plant Science volume 165 pages 85-93 and Terashima et al. 1998. Physiologica Plantarum volume 103 page 295–303.
Evangelina,
Thanks for your so complete answer! I'll see also the references.
Best regards
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