My system contains 1.8 ml buffer (pH 7.6, Tris sulfate), 1.0 ml 3mM p-NPA (para-nitrophenyl acetate) and 0.2ml enzyme solution. Change in absorbance is recorded at 25 degree.
The way to measure the extinction coefficient of any substance is simply to measure the absorbance of a solution of the substance with a known concentration and path length. According to Beer's Law, the extinction coefficient is the absorbance divided by the concentration and the path length. The extinction coefficient can be measured this way at any wavelength you choose, but it is usually done at the wavelength at which the absorbance is maximal.
When using the substance as the substrate of an enzyme and measuring its consumption by the loss of its absorbance, you should also consider the absorbance of the product. You may want to choose to monitor the wavelength at which the difference between the substrate and product absorbances is maximal.
Thank you adam for your promote reply.
Now, I want to know, which wavelength should i measure absorbance, 348nm or 410nm?
My equation is as attached.
The same equation could be used for either wavelength. If you make the measurement at 348 nm, use the 348 nm millimolar extinction coefficient in the denominator. If you use 410 nm, use the 410 nm millimolar extinction coefficient in the denominator and the 410 nm measurements in the numerator.
Hello everyone,
I am looking for a lipolytic activity in different cell fractions using a chromophore substrate (50µL) on microplate reader 96 well:
Vs is the volume of my cell fraction wich contain the enzyme =50µL and Vt is the total volume of enzyme reaction = 300µL
1- I have measured the absorbance (Abs) of the product : Abs Vs time (min) " I have an absorbance for each 5 min"
2- I have prepared a standard curve : Concentration of product (µmol/L) Vs Abs
3- Then I have calculated the amount of product released (µmol/L) by the regression using equation of standard curve: Concentration of product released (µmol/L) Vs Time (min),
I found many propositions to how we calculate the Enzyme activity; my question is what should i use to calculate Enzymatic activity (U/L = µmol/L/min) ?
A - U/L= Vt/Vs * 106/ ε.l * Δ Abs/Δt *DF : (ε is coeficient of extinction (M-1.Cm-1), Vt=300µL, Vs=50µL ....
- For ε, I found the table attached, but for my experiment, I measured the Abs at 415nm at pH7; Can I use it for my calculations?
- l =?? i don't know if I can use 1 Cm because I used a microplate 96 well for my reaction not a cuve.
- DF factor of dilution, I don't know it represent what? I used 50µL of substrate + 50µL of cell fraction(wich contain enzyme) + 200µL of Buffer.
B - Or just divide each Concentration (µmol/L) by Time (min) ? Δ Concentration/Δt * Vt/Vs
I want to compare the Enzymatic activity of two Cell fractions, how should I do? Can I choose a delta of concentration/delta of time (the same points of time for two Cell fraction) ?
Thank you for your help.
You do not need to employ the extinction coefficient at all if you have prepared a standard curve with the same sample volume in the same type of plate. Therefore, you do not have to take into consideration the path length.
(Incidentally, the slope of the standard curve is the extinction coefficient for that path length. You will sometimes see in papers the extinction coefficient for assays done in microplates written without the path length dimension because they have been measured this way. You can compare the slope of your standard curve to the published extinction coefficient if you want to measure the actual path length, although this is not usually necessary.)
The dilution factor is the total reaction volume divided by the volume of extract used: 300/50 = 6.
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