Hi,
I have a question about how to calculate the enzyme activity from absorbance. I have absorbance values for different concentrations of my samples at different times. As far as I understood, I should plot the time against the absorbance. I would have a series of curves corresponding to the different concentrations of my samples. The slope of these curves correspond to the enzymatic activity..is that right?
Thanks
Hi there,
The slope of the curve absorbance=f(t) is actually velocity of the reaction expressed as variation of absorbance per time unit (then velocity unit may be converted into mole/min if you know the relationship between absorbance and the concentration of product (ie. molar epsilon) and the volume of the assay). Activity will be velocity divided by the amount of enzyme present in the reaction assay (either mL of enzyme stock solution or preferably mg of protein).
What are your samples? Are they products of substrates converted by the action of your enzyme? Was one unity of your enzyme activity defined in the literature?
You can calculate the activity of your enzyme from absorbance. But one unity of your enzyme activity in relation with the amount or absorbance of the substrate or the product should be defined clearly. For example, for lipase activity assay, one unit of enzyme activity was defined as the amount that liberates 1 micromole of p-nitrophenol per minute under standard assay conditions.
You can check the materials and methods section of the paper published on lipase. Here is the link to access the paper. I hope you will get some helpful idea to apply for your experiment.
https://www.researchgate.net/publication/233579090_Increased_enantioselectivity_of_lipase_in_the_transesterification_of_DL--3-phenyllactic_acid_in_ionic_liquids
Article Increased enantioselectivity of lipase in the transesterific...
Hi there,
The slope of the curve absorbance=f(t) is actually velocity of the reaction expressed as variation of absorbance per time unit (then velocity unit may be converted into mole/min if you know the relationship between absorbance and the concentration of product (ie. molar epsilon) and the volume of the assay). Activity will be velocity divided by the amount of enzyme present in the reaction assay (either mL of enzyme stock solution or preferably mg of protein).
Dear Silvia.
I agree with Dominique, but you should check if the wavelenght that you are mesuring is related only to the product. Otherwise you can not relate it exactly to the activity. Note that the wavelenght of the reactant is not a valid option since it can be decomposed to other products through reactions that you are not studying.
Best regards.
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