Hi there,

Initial velocity may be reported to the volume unit of the enzyme sample you have used for the assay by calculating v0/volume of enzyme sample used in the assay (you may need to apply a dilution factor if needed). You get enzyme activity of the fraction expressed in µmol/L/min/mL of enzyme sample. Knowing the total volume of the enzyme sample, you may calculate the total activity in the sample by multiplying enzyme activity by the volume of the enzyme sample. If you do this for the different cellular fractions you have, you may compare the actual activity content of these different fractions.

Hello,

Thank you for your answer,

So, if I understood well what you noticed Sir, to calculate the total enzyme activity; I should do it like this: U/L= ΔC/Δt *Vs. Right?

which Vs is the volume of the enzyme solution taken (=volume of the cell fraction that contain enzyme that I am looking for)

In the essay, I used 50µL of substrate + 250µL of solution Buffer + 50µL of cell fraction ( Vs )which contain the enzyme = Vt 300µL. In this case, sould I apply a dilution factor?

The intial velocity represented by : Vo=ΔC/Δt (µmol/L/min). Please, why you divided it by volume of enzyme sample ?

Hi Rebiha Adjout, when you are purifying an enzyme it is helpful to calculate specific activity. That is umol of substrate turned over (in the linear range) per ug of protein you use for the assay.

The higher specific activity you have, the more pure your enzyme is. Of course to determine purity you should do an SDS-PAGE.

Hi,

I didn't do a purification of enzyme.when I said "fraction" it means Cell fractions not fraction after purification.

I have mesured the Concentration of product released Vs time of different cell fractions like Whole cell, intracellular...and to compare between them I ask how I can do it using Total enzyme activity?

Hi, you are doing enzyme purification by measuring enzyme activities in differential fractionation of cells.

Again, specific activity will give you activity/ug of protein. Total enzymatic activity is generally not a useful parameter, but you could multiply total protein with specific activity. Bear in mind that this total activity only applies to the substrate you are measuring. Other substrates can have different Km and Vmax.

Hi again,

If your intention is to compare total enzyme activity in different fractions, you have at some point to make the calculation of the theoritical velocity you would get if engaging the full fraction in the assay (total activity). To do so you first have to express the initial velocity you have actually measured toward the enzyme volume unit (if volume of fraction used in the assay is 50µL then you calculate v0/50µL) and then the total activity of the fraction is (v0/50)*total volume of the fraction expressed in µL. In your case there is no dilution factor to consider as you haven't diluted the fraction prior running the assay neither total volume of the assay.

Thank you all for your answers ! Iget it

To calculate total enzyme activity in different cellular fractions you need to determine initial velocity in a particular volume of enzyme , in your case it is 50 microlitre. then multiply this value with total volume of the cellular fraction i.e in your case units/50microlitre multiplied by total volume of cellular fraction..