I performed hydrogen peroxide radical scavenging assay. After increasing the concentration of sample, absorbance was also increased. Even the absorbance of control is lower than the sample. If anybody has an idea please suggest the reason behind this and suggest any modification in the protocol.
I used two different protocols for the experiment but got increasing order of absorbance both times.
2mM H2O2 solution (30%), and 50 mM phosphate buffer (pH 7.4)
40 mM H2O2 solution (30%), and 50 mM phosphate buffer (pH 7.4)
The concentration of ascorbic acid is 10 micrograms/ml to 100 microgram/ml.
Thanks
Dear Preety,
as far as I know, due to its chemistry, ascorbic acid mainly detoxyfies radicals by donating an electron to them therefore cannot detoxify H2O2, which is not a radical. If you like have a look at these articles:
Article A theoretical study of ascorbic acid oxidation and HOO / O 2...
Article Ascorbate is outstanding antioxidant in human plasma
Article Vitamin C: Newer Insights into Its Biochemical Functions
Article Functions of ROS in Macrophages and Antimicrobial Immunity
But perhaps a chemist (and not a biologist) can give any advice here, since a ROS racevenger is not automatically an antioxidant, and sometimes one ROS subspecies is converted into another.
All the best and stay healthy,
Marc
Are you reading the absorbance at which wavelength?
Note that hydrogen peroxide is usually read at 240 nm and ascorbic acid does absorb light at this wavelength (although it is not its λmax).
Please suggest an alternate standard antioxidant compound that I can use for this assay.
Typically absorbance increases with increasing sample concentration (Lambert-Beer law).
Mirosław Grzesik Exactly Sir, the absorbance got increased with increasing sample concentration. But in the case of antioxidant assays like DPPH & ABTS, the absorbance of the sample decreased with increasing sample concentration and results in increasing % inhibition of the sample. So, according to the formula = Abs (Control)- Abs (Sample)/Abs (Control)X 100 the absorbance of the sample should decrease with increasing concentration of the sample.
Preety Rohilla, Of course, in the case of additional effects or accompanying reactions, the Lambert and Beer law will not apply. My answer was general, but thank you for your explanations.
Preety Rohilla, You find the answer in this paper,
Ruch et al. - 1989 - Prevention of cytotoxicity and inhibition of intercellular
communication by antioxidant catechins isolated from Chinese
green tea.
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