I am studying the activities of Laccase enzyme in crude extract of P. chrospordium fungi Using ABTS. I cannot observe change in absorbance at 420nm or other wavelength. I used different protocols and different ways with all possible options (I attached one of the protocols). Furthermore, according to the protocol, the molar adsorptivity for radical ABTS is 36000 M -1 cm-1, Therefore, if we use 0.5 mM of ABTS in a 1*1 cm2 cell we should see finally 18 unit increase in absorption (A). But the range of our instrument (Varian-Carry 200) is 0-10. So how we can use this instrument for laccase assay?
You do not need to convert 100% of the ABTS to product. It is usual to set up an assay to convert less than 10% of substrate to product in an enzyme reaction so that the initial rate of the reaction can be measured. In this case, 10% conversion would result in an absorbance change of 1.8 absorbance units, which is a very large signal, much larger than necessary. Even 1/10 of that signal would be plenty.
It is not realistic to measure absorbances much larger than about 3 (99.9% of light absorbed), because the amount of light remaining becomes too small to measure, even if the instrument software claims to be able to measure absorbance up to 10.
What is the background absorbance you measure in your assay with no enzyme? Is it low enough to allow a further absorbance increase to be measured?
The background absorbance is less than 0.1. But When I add enzyme solution, since it has other colorful compounds, it goes up to 0.4.
Agree with Adam's comment(s). Measure the A420nm of the enzyme solution only and use that as a background reference. For enhanced sensitivity, use the double beam nature of the spectrophotometer and place the enzyme solution in the ref beam and the assay cocktail in the sample beam. The difference in OD detected should be related to the converted substrate assuming no spectral interference from the sample itself at 420nm.
Thanks, I did the experiment again and I faced another problem. The radical ABTS is not stable. For example, it takes 5 min to reach the maximum amount of absorbance and then it comes back to baseline. It mean that there is a reaction that consumes radical. Therefore how we can measure the true amount of absorbance, when there is another reaction consuming the the radicals?
Hallo, perhaps you are facing the presence of a reductase, converting back the ABTS oxidation product to its reduced state. This implies your laccase is unable to convert again ABTS to the colored radical. This is reasonable if the assay solution is static, and no atmospheric oxygen is allowed to dissolve rapidly into the solution. You could easily check the reductase hypothesis by gently shaking your assay solution: if the hypothesis is correct, the green color should rapidly arise. Let us know, please...
Dear Enrico
I also used potassium persulfate for comparison. to my surprise, after 5 minutes the color was completely disappeared which means that even without reductase the radicals are not stable.
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