I want to study the activity of laccase. All literature are using extinction coefficient values (epsilon) for laccase substrates i.e: syringaldazine (SGZ), dimethoxyphenol (2,6-DMP),...
Despite the fact that it using them is easier than preparing a standard curve, each paper uses a different epsilon value for the same substrate.
Has any one prepared standard curves for the mentioned substrates?
Is it possible?
or should I follow any published value for my work?
Dear Sherine,
The best method for measuring the enzyme activity is to prepare the standard curve of its substrate or its product. I have prepared ABTS standard curve for measuring laccase activity.
In principle, the epsilon values should not change from one paper to another, but they could change if the product changes its absorption spectra with change in solvent, pH, salt effects etc. Also, laccase produces polymeric materials whose concentrations would vary with reaction period. It is best to perform assays of your own with well defined reaction conditions. It takes only a couple of hours to have standard activity graphs.
Mina Karimi-Avargani Would you please elaborate how have you done it? Did you use Trolox as your standard?
Nitin Fadnavis Thank you for your answer. How can I produce my own extinction coefficient value?
The procedure is straight forward. Remember that the wavelength of monitoring activity is substrate dependent. For example, ABTS 418 nm, o-dianisidine 460 nm, guaiacol 470 nm, 2,6-DMP 468 nm, SGZ 525 nm etc. All parameters (except substrate conc.) should be constant: the pH, buffer concentration (usually 0.05 M to 0.1 M), enzyme concentration (e.g. 1 mg/mL), reaction volume (e.g. 2 mL), temperature etc. It is best to make a stock solution of substrate of your choice (I recommend ABTS, Mol wt 514) 0.3 mM by dissolving 15.4 mg in 100 mL of buffer.
Also prepare a 10 mL stock solution of laccase in buffer of same pH (1mg/mL) and distribute 2 mL each into 4 to 5 cuvettes. Adjust the wavelength to 418 nm, make zero correction and add 10, 20, 30, 40 microlit of ABTS solution to the cuvette respectively, mix and measure absorbance after 10 min. or till the absorbance remains constant for at least 10 min. Make a plot of final absorbance vs ABTS conc. in cuvette (if you have taken 10 microlit of 0.3 mM ABTS in 2 mL enzyme solution in cuvette, the conc. of ABTS in cuvette would be (0.01 x 0.0003)/2.01 = 1.492 micromolar. If the path length is 1 cm, and observed absorbance is 0.054, the extinction coefficient would be (0.054/1.492E-6) = 36,012 M-1cm-1. The slope would give you the value of extinction coefficient. This is just an example. You can choose your assay substrate available in your lab. The pH-optimum also depends on the source of laccase so you have to chose buffer of appropriate pH.
@Nitin Fadnavis
Thank you so much. That's very elaborative.
Dear Sherine,
1. Prepare different concentration (min 5 different concentrations)of substrate (ABTS).
2. Plot the standard graph of substrate (Adsorption - Concentration).
3. According to the adsorption of enzyme and the standard curve, you can find the concentration of unreacted substrate.
4. From the initial concentration of substrate and unreacted substrate, you can calculate the consumed substrate.
The enzyme activity is calculated according to the following formula:
Enzyme Activity(μmol/min ml) or (U/ml) = (Consumed Substrate) ×Total Reaction Volume / (Reaction time (min)) × (Enzyme volume(ml))
Mina Karimi-Avargani could you please further explain point no. 4? How can I calculate the consumed substrate value from the unconsumed substrate value?
and shouldn't the ABTS wavelength be different from the oxidation product of reaction? Thank you in advance
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