I carried out laccase assay using ABTS. Initially, the ABTS was green when I dissolved it in the assay solution but by the end of the assay (10 minutes), the green colour was gone and the solution was light brown. I don't know if the brown color is the reflection of the supernatant from lignin media I used as crude enzyme solution The absorbance reading (420nm) also decreased instead of increasing.
Is it possible for ABTS to be reduced or bleached in the assay?
I am certain that you did not use pure compounds. ABTS during laccase assay has a tendency to react with other low molecular weight compounds. Moreover, it is a laccase mediator that oxidizes low molecular weight substrates such as non-phenolic lignin components with high redox potentials like veratrylalcohol and lignin dimers such as 1-(3,4-dimethoxyphenyl)-2-phenoxyethane-1,2-diol. It is possible for secondary reactions to result in decolorization.
Maurice Ekpenyong Thanks for your answer sir, I will try the assay again, although I have experienced this two consecutive times now. As for the purity of my compounds, I only used the supernatant from my culture (lignin minimal salt media) the other thing I added was just the buffer and ABTS as the protocol requires. That's why I am confused about the orange-brown colour of the assay solution at the end of the assay. Hopefully, I will give updates in case get a positive result.
Usually, the green color developed through the oxidation of ABTS by laccases should be monitored through one minute reaction. Through this one minute reaction you will determine the reaction kinetics from zero time and one min reaction absorbance. This usually depending on the activity of your enzyme, which means when the activity is high you should get your readings in a short time (1 min), but if it needs more time due to the lower activity you can prolong the time. I think the change in the developed color is due to the loss of appropriate time point to get the absorbance value and this may be due to the higher activity. As a solution, you can try one of these probabilities: 1) use diluted enzyme; and 2) try to short your reaction time. May be you have a look at this article.
Othman, A.M., Elsayed, M.A., Elshafei, A.M., Hassan, M.M., 2018. Purification and biochemical characterization of two isolated laccase isoforms from Agaricus bisporus CU13 and their potency in dye decolorization. International Journal of Biological Macromolecules 113, 1142–1148. https://doi.org/10.1016/j.ijbiomac.2018.03.043. Good luck
Abdelmageed Othman Thanks for your suggestions. I will put them into consideration and hopefully let you know the outcome if I get good results. Thank you
Dear Friend,
The color of the ABTS solution must be green, but as soon as adding the enzyme solution the color of sample changes, because ABTS as the substrate of laccase reacts with enzyme and the color of the reaction product is different from the color of ABTS. Moreover, by passing of time the amount of absorption decreases due to consumption of substrate (ABTS). Therefore, there is not anything wrong in your experiment. It is only necessary to incubate ABTS solution after adding enzyme during a specified time. The length of time depends on enzyme activity. If it is pure enzyme, 2-5 (min) is enough. It is necessary to measure the absorption for all samples after passing a specific time period.
Mina Karimi-Avargani Thanks for your contribution, I will work on the timing of my assay
Presently I am working on bacterial laccase mediated delignification of lignocellulosic biomass. In regards to that, the isolated bacteria was grown in Luria Broth and the laccase assay was carried out by monitoring the absorbance at 420 nm up to 7 days post-inoculation. The reaction mixture contained- 510ul buffer (citrate phosphate buffer, pH 5), 300 ul of enzyme and 90ul of ABTS (0.5mM).For the assay the enzyme was diluted 5 times. The absorbance was found in the range of 0.2-0.9 (1st Day to 7th Day). However, the light green color of the ABTS disappeared once added to the reaction mixture (diluted enzyme and the buffer). As per literature, the color of the reaction mixture should change to green once ABTS is added. The color of the supernatant was also reddish brown on 7th Day. So the day wise absorbance might be the reflection of the supernatant. Also I am planning to check for the intracellular laccase production. Please suggest the required changes so that it works out. Thanks in advance.
N.B. - The bacterial colonies was found to be reddish brown after a certain incubation period on guaiacol plates and so was considered laccase positive.
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