Sir Thank you for your reply.

Sir 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.

Sir so as soon as I add enzyme then I have to take O.D. This gives 0 absorbance.

Thank you for reply.

Hi, I will tell you what to do.. Take abs. at zero time i.e. before heating your sample. make it zero. (auto zero is available in any UV spectrophotometer ). Then take readings at regular intervals. from the standard curve find out the concentration of Maltose formed from starch.

Secondly 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.

Hi! You could try to draw the diagram where X axis is time after adding the enzyme and Y axis is Abs, and if you could approximate the dots with line with low error (you must have at least 3 dots but it's better to have more) you dont need Abs at 0 min to calculate enzyme activity. (It will be linear if enzyme do not inactivated during reaction and substrate consumption is low)

Molar 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

Gentleman just do the following-

1. 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...

Hi everyone, Thank you for your suggestions.

I give you the form for calculating the enzyme activity. There are 2 kinds of formular for calculating base on the ratio of sample and DNS solution.

Dear Nguyen, Can you suggest how to calculate the exact activity with some reference?