# I have few doubts in calculating enzyme activity.

I have plotted standard graph using Maltose(0.5mM to 3mM)

now i have to calculate enzyme activity of amylase from saliva using starch (5%)as substrate with DNS reagent (dinitro salicylic acid). I have taken O.D for 3 min( i did not take O.D at 0 min.)

so my question is for calculating Molar absorption coefficient do i need to take concentration from each min. (back calculated from O.D) Or concentration obtained from the difference of O.D (O.D of 1st min - O.D of 2nd min)

I would like to know the formula to calculate enzyme activity.

I have found on formula for enzyme activity,

delta Absorbance X total enzyme assay volume X dilution factor

U/ml= --------------------------------------------------------------------------------------

time of assay in min. X Molar absorption coefficient X volume of enzyme extract

I have doubts in this also,

total enzyme assay volume - means what do i need to take? is it only substrate and enzyme volume or do i need to include volume of DNS reagent added and water that i added before taking O.D.

Delta absorbance - means is it O.D of each min or O.D difference of 1st and 2nd min

time of assay in min. - means is it 1min or 3min

now i have to calculate enzyme activity of amylase from saliva using starch (5%)as substrate with DNS reagent (dinitro salicylic acid). I have taken O.D for 3 min( i did not take O.D at 0 min.)

so my question is for calculating Molar absorption coefficient do i need to take concentration from each min. (back calculated from O.D) Or concentration obtained from the difference of O.D (O.D of 1st min - O.D of 2nd min)

I would like to know the formula to calculate enzyme activity.

I have found on formula for enzyme activity,

delta Absorbance X total enzyme assay volume X dilution factor

U/ml= --------------------------------------------------------------------------------------

time of assay in min. X Molar absorption coefficient X volume of enzyme extract

I have doubts in this also,

total enzyme assay volume - means what do i need to take? is it only substrate and enzyme volume or do i need to include volume of DNS reagent added and water that i added before taking O.D.

Delta absorbance - means is it O.D of each min or O.D difference of 1st and 2nd min

time of assay in min. - means is it 1min or 3min

## All Answers (8)

DeletedIf take readings from 3 min your enzyme already produces products by reacting to the substrate. So as you follow it will give errors..

Thejashree Sarepalli· Institute of Bioinformatics and Applied BiotechnologySir after adding the enzyme I incubated at 37 degree C for 5 min, During that 5 min, I have collected 300ul of solution mix(substrate+enzyme)after 1 min and transfered to 300ul DNS reagent. Like this I have collected solution mix for every 1 min interval. for 3 min so what ever product (maltose) it will react with DNS reagent and forms colored substance thus stops the reaction. This is what I have understood. If I am wrong please correct me.

Thejashree Sarepalli· Institute of Bioinformatics and Applied BiotechnologyThank you for reply.

Sanket Jadhav· Curtin UniversitySecondly Assay volume consists of only enzyme plus substrate and buffer if any.

Color of DNSA might be very high so u have to dilute it. Include that dilution factor there. Activity formula which u have given is clear nw. So directly use it to get ur answer.

Vladimir BarkhatovMolar absorption coeffitient {M-1*cm-1}=abs/([Maltose{M}]*(optical path{cm}))

and should be similar for all dots with Maltose

enzyme activity = velocity {mkM/(sec*(mkl of enzyme))} = (delta Abs)/(delta time{sec})/(M.a.c.{M-1*cm-1})*(600{mkl}/(volume of enzyme extract per 300 mkl of solution mix{mkl}))*10^6

Shiv Kumar Meena· National Institute of Immunology1. Calculate plot from standard of Maltose (uM to mM range) ie. 0.uM to 500uM and 0.5mM to 3mM

2. Use the slope for calculating the concentration of product (Assay of your Enzyme)

3. Now divide OD at each time point with sloe, you have the concentration of product (Maltose)

4. divide the product conc with time taken for assay

5. You have /min or /unit time product (Rate

6. Plot it at Sigma plot and fit into apt equation okay.......

you will get activity profile of your enzyme...

Thejashree Sarepalli· Institute of Bioinformatics and Applied BiotechnologyNguyen dang hai dang· Sangmyung UniversityCan you help by adding an answer?