One simple method is to use the UV absorption peak at 240 nm, A commonly used assay for catalase activity uses H2O2 in buffer and records the decrease in OD vs time,

I have not been responsible for extracting H2O2 from anything, but you might be able to measure it =/- catalase, Just a thought

Best regards

Rick Tullis

CSO

Aethlon Medical

Dear Richard,

Thanks a lot. That is a good idea. We have been measuring catalase activity by recording decrease in OD vs time (which is due to decrease in H2O2 level).

However, proteins, nucleic acids, phenols also absorb at these wavelength (although their absorption peaks are at 215-230/280, 260 and 270, respectively). If we have pure samples of each of them, then it will not be difficult (as there is nothing to interfere with).

I certainly believe that measuring H2O2 in presence and absence of catalase will be a good solution, in case if we extract H2O2 in a medium that will not inhibit catalase. In case we extract in 5% TCA, we need to neutralize before the same can be used determining H2O2 level. We shall try and get back to you.

best regards

Pardha

Sir,

       Maybe this paper may help.

Dear Sarang,

Thank you. We used this method (you may see our paper in Plant Molecular Biology), but it is too complicated. Major issues are use of charcoal and then adjusting pH.

Dear Prof. Pardha-Saradhi,

You can use the following protocol:

Crush 0.1 g of tissue (leaf, root or shoot) (previously collected under ice cold conditions) in 0.1 % tri-chloro-acetic acid (TCA) and homogenize it at 4 °C. After centrifugation at 10 000 g for 15 min, keep the supernatant in dark for 1 h after mixing with phosphate buffer (10 mM, pH 7.0) and potassium iodide (1 M) (Add in the ratio 0.5 ml: 0.5 ml: 1 ml). Record the absorbance of the resulting solution  at 390 nm.

All the steps were performed at 4 °C except absorbance measurement.

This is the simplest method for detection of H2O2. We have been doing it for last 5-6 years from different plant parts.

Also see the following publications of our lab (attached as pdfs):

(1) J Agron Crop Sci (2014) 200; 273–289

(2) IJBB 50 (2) (2013) 139-149

I hope this helps.

Dear Dr. Mishra,

Thanks a lot. Can you let me know, (i) if 0.1% TCA is sufficient to pellet down all proteins; and (ii) are you using 10 mM phosphate buffer to neutralize the reaction mixture?

Respected Prof. P. Pardha-Saradhi,

Many thanks for your message. I am a PhD scholar, about to submit my thesis.

(1) Yes 0.1% TCA is sufficient to precipitate proteins in my plant samples (monocot leaves). If complete precipitation is not there, we can use 0.5% TCA for complete precipitation.

(2) Yes we are using 10 mM Phosphate buffer for neutralizing the mixture.

Warm regards,

Amit

Dear Amit,

Thank you for attending to my queries.

Good Luck

Hi! Not sure if this will work for plant extracts, but did you try the Iodine/Starch or Titanium oxalate methods? The first is giving very strong blue color ( 39450 mol-1*cm-1*L at 570 nm), while the second is yellow (about 400nm).

Regards,

Dumitru.

Dear Dr. Dumitru Sirbu,

Thanks a lot. We have tried both the protocols.

Way after the discussions of 2015 for sure but. I recently looked up the papers of Dr Mishra (thanks) and found the original method is attributed to Sergiev and co-workers (2001). Indeed others had cited H202/KI method to the same author (Siegiev et al. 1997) but this was in a book chapter and not available online. The 2001 paper with full protocol appears as [[Alexieva, V., Sergiev, I., Mapelli, S. and Karanov, E., 2001. The effect of drought and ultraviolet radiation on growth and stress markers in pea and wheat. Plant, Cell & Environment, 24(12), pp.1337-1344.]]  Great news is the original paper is available on Research Gate

Article The effect of drought and ultraviolet radiation on growth an...

Thanks Prof. Richard for the encouragement. The protocols available in the literature always work, if we follow exactly. But some minor modifications may be required depending upon the experimental material and conditions. 

Hello Amit Kumar Mishra ,

In your answer you said that 10 mM KI is added to neutralize the solution, however the reaction need to be done in acidic medium ... this is confusing!!

Dear Dr. Walid,

I didn't said KI...it's the phosphate buffer. Please check again the conversation. In the protocol (Alexiva et al. 2001), we generally use TCA (Trichloroacetic acid), which itself is an acid, so no need to make the mixture more acidic. In our group, we are following this protocol from past several years and it worked for lot of plants used as experimental material.

Regards,

Amit

Dear Dr. Mishra,

yes sorry it was a typing mistake ... yes I know the protocol and I followed it but again when I returned back to publication, it is said that the reaction between KI and hydrogen peroxide goes in acidic medium ... so I am confused why then to neutralize??!! ... well, I did a standard curve for hydrogen peroxide in 1% TCA and I did not neutralize and I have got a nice straight line with R almost equals 1 ... that is why I am confused why all literature do neutralization?

Dear Dr. Walid,

What you said is absolutely correct! There is no need to neutralize the mixture. The protocol also works without addition of phosphate buffer. As I stated earlier in the conversation, every protocol need minor modifications. As per my point of view, physiologically, every reaction performs best at neutral pH (pH 7.0), so addition of phosphate buffer neutralize the reaction (chemically) in the given protocol (Alexiva et al. 2001) for H2O2 estimation.

Regards,

Amit

how do I purify hydrogen peroxide after extracting it from plants?

To the best of my knowledge, it is impossible to purify hydrogen peroxide from plants or plant extracts. By the way, why do you want to purify hydrogen peroxide "after extracting from plants"?

I want to know if it can it can produce commercially quantity just the way lipid is produced from algae

It is possible to purify and concentrate majority of organic bio-molecules [in particular proteins, lipids, polysaccharides, RNA, DNA, compatible solutes, phenolic compounds, organic acids, amino acids etc.]. However, to the best of my knowledge it is not possible to purify reactive oxygen species like hydrogen peroxide. We can quantify, but we can't purify ROS. We can enhance their generation by imposing stress.

ok then, thank you Prof. Pardha-Saradhi

Walid Abuelsoud I know this is a thread from a while back but I am having trouble with determining hydrogen peroxide in sweet potato roots. I cannot even figure out the standard curve. Would you care to share your protocol? I have 30% hydrogen peroxide that am diluting to 20mM using distilled water for the standard curve. I diluted it further with TCA (0.1%) to make concentrations ranging from 0-20mM. Then I used 0.5ML of this sample (standard sample), 0.5mL (10mM pH 7) phosphate buffer and 1ml 1M Potassium iodide, incubate for an hour and then measured at 390 but it failed. What am I doing wrong? It has not worked for my actual sample either