I have to dilute the commercial Laccase and HRP (Horseradish Peroxidase) in 4 Unit/mL and 6 Unit/mL. How to dilute these commercial enzyme? As per different literature, ABTS oxidation per minute is to be measured (absorbance) to determine the U/mL of enzyme activity.
In this case, the continuous spectrophotometric assay is suggested to determine the U/mL of enzyme activity. How to run this continuous OD assay? Please help me to run this time depended assay of ABTS for enzyme activity.
Hi there,
Absorbance at 420nm is increasing with ABTS oxydization so if you are equipped with a spectrophotometer with a kinetic mode you just have to monitor the reaction running in a cuvette. For accurate measurements, you have to get a linear curve (initial velocity ) on the ealy part of the curve (slope being the initial velocity). Enzyme concentration has to be adjusted in order to get this linear signal on decent laps of time to be able to calculate the slope. Then this initial velocity expressed in variation of OD per time unit can be converted in M/min (using Beer's law and epsilon of 34000M-1.cm-1 and l=1cm) then knowing the volume of the reaction you can convert it into mol/time unit by multyplying the latter result by the volume expressed in litre. Then knowing the volume of enzyme present in the assay you can calculate activity by dividing the result by this volume in mL. Then convert it in U/mL (one unit being 1nmol per minute). And finally take into account the initial dilution of your enzyme to get the activity of the enzyme sample (multiply by the dilution factor).
Hello,
Sigma has a quite detailed protocol on their webpage. Here is the link:
https://www.sigmaaldrich.com/technical-documents/protocols/biology/enzymatic-assay-of-peroxidase-abts-as-substrate.html
Good luck!
Thanks dear Dominique Liger and Gisela Beutner for your contribution regarding this. I found the protocol now, let me try with these protocol.
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