This link must help you
http://www.sigmaaldrich.com/technical-documents/protocols/biology/enzymatic-assay-of-laccase.html
For determination of laccase activities you mix your enzyme solution with an ABTS stock solution at a defined pH (use a buffer solution like e.g. McIlvaine phosphate citrate buffer). ABTS concentration of the mixture should be 0.2 mM. Then you measure absorbance over time at 420 nm.
The enzymatic units (U) defined as the amount of enzyme transforming 1 µmol of substrate per minute of your enzyme stock solution per liter are then calculated by the following formula:
U/L = (∆E×Vt)/(ε×d×Vs)
with ∆E being the change in extinction of light [min-1] at 420 nm, ε being the molar absorption coefficient of ABTS [M-1 cm-1] (this you have either to determine yourself or use a value from literature, be aware that the coefficient is pH dependent), d being the layer thickness [cm] in your cell that the light has to pass, Vt is the total volume you measured and Vs is the volume of the enzyme stock solution you added to the ABTS stock solution.
Dear Christopher Andreas Gasser,
Thank you for your post regarding determination of laccase activity.If possible kindly mention the author of the paper or source of the protocol.
dear Christoph Andreas Gasser
I may be wrong, but it looks like your calculations lead to MU/L instead of U/L
Dear Pallavi Biswas,
one publication using this protocol is for example:
Zimmermann et al. (2011) Sorption-assisted surface conjugation: a way to stabilize laccase enzyme. Applied Microbiology and Biotechnology 92:169
Dear Simone Becarelli,
You are correct! If you use the units I provided above you end up with (mol/min)/L. To arrive at the U (defined as µmol of substrate per minute) you have to divide by 10^6.
dear Pallavi Biswas
are you sure that you don't have to multiply for 10^6 instead of divide?
1 mol =1000000 µmol , you make the substitution and here you go.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biochemical Techniques
- Analytical Chemistry
- pH