Does this formula accepted? Or they are other formula that are more widely used and accepted?
Units (U/ml)=
(microgram of glucose released) X (total assay volume) X dilution factor)
/ divided by
(volume of enzyme used) X (volume in cuvette) X (incubation time)
You are almost right, remember enzyme activity is given in:
mole of substrate degraded or product formed / (unit time*mg of enzyme) also note that you use the appropriate prefix for mole, it can be micromole, nanomole or picomoles. Time can be in sec, min or hours. Also personally I prefer to account for dilution factor in the protein concentration, whatever you do please be careful not to account for things twice.
so your equation becomes
[concentration of glucose released (molar)] X (total volume in cuvette) / divided by
(final concentration of enzyme) X (volume of enzyme) X incubation time
Hopes this helps
Marc
The volume in the cuvette is necessary to determine the actual number of moles of glucose that your system is releasing. Most people first obtain a molar value of their analyte that needs to be converted to mole for obtaining specific activity numbers
Hope this helps
Marc
I have already tried to use 1ml and 2ml of volume of sample but the absorbance reading does not change by volume. Wouldn't be wrong to include total volume of assay in cuvetter in the formula?
I got same reading of absorbance even the volume of assay in the cuvette varies.
Anyone could explain my confusion?
Remember the light beam from the spectrophotometer only cover a small portion of your sample size
Hope this helps
Marc
One unit of enzyme activity is the amount of product formed (or substrate used) per unit time. The SI unit (U) is 1 micromole of product per min (or 1 nanomole of product per min = 1 mU). To calculate the specific activity of your biological sample divide the enzyme units per unit volume by the the protein concentration per unit volume to obtain U/mg (note that the volume term cancels out whether U/L and mg/L or U/ml and mg/ml, etc).
With regard to the absorbance reading it is the pathlength of the cuvette that is fixed so you should obtain the same absorbance if you carry out the assay in 1 ml or 1 L, provided the final concentration of enzyme is the same (e.g. 1 mg in a 1 ml assay or 1,000 mg in a 1 L assay).
Hope that helps resolve your confusion.
Alan
Absorbance is proportional to concentration and pathlength. Volume of sample in cuvette doesnt alter absorbance. However, volume of enzyme, concentration of product matters.
Hi there,
use the same cuvette that you used in the standard curve.
Hello, may i asking the total assay volume means?
For example,
1) The solution contains 40 mg of soluble starch in 1.0ml of 0.1M sodium phosphate buffer
2) then, the solution added 0.5 ml of CGTase (enzyme) and incubated at 45-degree celsius for 10 minutes.
3) after that, 3 ml of 30 mM NaoH is added followed by 0.5 ml of 0.02% (w/v) phenolphthalein and stood at room temperature for 15 minutes
4) colour intensity was measured at 550nm
Total assay volume = 1.0ml (0.1M sodium phosphate) + 0.5 ml (CGTase) + 3 ml (30 mM NaoH) + 0.5 ml (phenolphthalein) ?
This book might resolve your questions.?..
https://books.google.com/books/about/A_Flexible_System_of_Enzymatic_Analysis.html?id=R23cNZbBByMC
Formula for specific activity= No. of units per mL/ concentration mg per mL
Enzyme Activity(μmol/min ml) or (U/ml) = (Consumed Substrate) (μmol /ml) ×Total Reaction Volume (ml) / (Reaction time (min)) × (Enzyme volume(ml))
Enzyme Activity(μmol/min ml) or (U/ml) = (Concentration of product of reaction) (μmol /ml) ×Total Reaction Volume (ml) / [(Reaction time (min)) × (Enzyme volume(ml))]
I was looking for the solution... thank you Sreenu Thupakula for the calculation.
Dear @Mina Karimi-Avargani could you give me the reference for the formula, please?
Dear all, the most conventional formula name is 'Michaelis–Menten formula', but most enzymes show deviation from it. Please check the following links and documents. My Regards
https://www.researchgate.net/post/how_to_calculate_enzyme_activity_from_absorbance
https://doi.org/10.1016/S0075-7535(08)70516-3
- Badges
- Science topic
- Similar topics
- Filing