I am running an experiment testing HRP-activity of Gquadruplex and hemin. Reaction works and ABTS turns blue and I can read a significant the absorbance change within seconds. But my problem is that the color turns back to yellow quickly. as if ABTS is reduced back to original form. Any ideas why?
H2O2 is not so much stable. It can happen that H2O2 decompose to H2O and O2.
but the problem is the reaction starts and tube mixture becomes blue but then it goes back to yellow. even if H2O2 was unstable there is no reason for blue ABTS+2 to go back to yellow ABTS.
ABTS has a charge 2- and is transparent, it does not have a color. 1e-oxidized ABTS has a bight green color. I do not understand what you actually see.
In my experiment ABTS enters the reaction with H2O2 to get oxidized and change its color from tranparent to green (or blue). Until here I have no problem. But within minutes the color starts to change back to transparent. But I need that the color is stable at least for an hour. why wud the color change back to transparent?
ABTS does not react with H2O2, but 1e-oxidized ABTS might react regenerating initial ABTS. H2O2 is not a good oxidant to make ABTS(-.) You can easily make 1e-oxidized ABTS stock solution using solid PbO2 as an oxidant under acidic conditions as described in my papers:
Methods Enzymol. 2002;359:366-79.
Evaluation of antioxidant activity using peroxynitrite as a source of radicals.
Geletii YV, Balavoine GG.
Russian Journal of Bioorganic Chemistry
November 2002, Volume 28, Issue 6, pp 501-514
The Determination of Total Concentration and Activity of Antioxidants in Foodstuffs
Yu. V. Geletii, G. G. A. Balavoine, O. N. Efimov, V. S. Kulikova
In you system in the excess of H2O2 ABTS might be oxidized by 2 -electrons. 2e-oxidized ABTS is unstable and decomposes to yellow colored products.
Thank you, Yurii. I only have H2O2 at the moment, but I will try with lower H2O2 concentration.
Hello Osman, I am curious if you had solved this issue. I am having a similar problem with yours. The assay was to apply diamine oxidase to metabolise histamine and meanwhile to generate hydrogen peroxide, and then ABTS and HRP were used to detect H2O2. The green color showed up in several seconds but turned to be colorless in several minutes. I was unsure whether hydrogen peroxide was generated or not.
In mycase lowering h2o2 Conc worked but I ended up staying away from ABTS and used fluorescent dyes. Let us know if if u notice any improvement.
I apparently used 100 times more concentrated then I should have. Simply dilute using double distilled water but bear in mind that at low conc H2O2 is way less stable so prepare from stock each time. Unfortunately that opens possibility to handling errors.
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