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Author Topic: Enzyme Activity  (Read 1244 times)

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ganesh2gig

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Enzyme Activity
« on: August 08, 2017, 01:17:20 AM »

Dear All ,

I just wanted to know the data interpretation I am doing to be  right or wrong regarding enzyme analysis .

Context: I work with  powdered samples having unknown concentration of enzyme like amylase , protease etc..   there is no requirement of specific activity to be declared all I need is  general activity / gram of sample .

Approach to finding out activity :  What I do is ,

1. Make a known dilution of the sample example- 100 mg in 100 ml buffer .
2. keep the substrate  volume constant and do analysis  with aliquots from step 1 - 0.1, 0.2 ,0.5 ….1 ml
3. Develop color using the reagent and take the reading .

I am aware in step 2, I am supposed to change the substrate concentration, but for the convenience sake   I am doing this way . ( By the way the data is always relative , right ?  as one should be constant and other can be a variable ) .

4. Plot a graph of OD  Vs Dilution , example 

100 dilution - 0.1
500 Dilution - 0.5
700 Dilution - 0.6

Now I know the rate of change for dilution  to OD decreases and nears a constant  , this is where the saturation is and I calculate my activity from that point i.e  the point where change in dilution does not change the OD considerably .
( Vmax is independent of substrate concentration )

Calculate all the necessary factors like dilution , time , M.W of end product and declare the result.

Just wanted to know if this is right , any inputs are more than welcome . 
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ganesh2gig

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Re: Enzyme Activity
« Reply #1 on: August 09, 2017, 01:11:02 AM »

Or should I look at the initial rate of the reaction ?
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Yggdrasil

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Re: Enzyme Activity
« Reply #2 on: August 09, 2017, 04:51:28 AM »

In your assay, are you monitoring the product or reactants by spectrophotometry?

Activity measurements are always rates, so you should be measuring multiple timepoints in your reactions and calculating the initial rate or reaction, not looking just at the endpoint.
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ganesh2gig

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Re: Enzyme Activity
« Reply #3 on: August 09, 2017, 06:41:18 PM »

Thanks for the reply .

I am measuring  the end product  of the enzymatic analysis . 

When you say multiple time points , does it mean I keep the substrate concentration constant and monitor the rate of the reaction at 10 ,20 ..30 minutes ?

From what I understand about initial rates is that the OD should be less and the dilution should be highest .
For example I will enumerate the data ,
2000 dilution = 0.179 OD
10,000 dilution = 0.190 OD
50,000 dilution = 0.192 OD
100,000 dilution = 0.052 OD

If I consider 0.052 OD the activity would be 23,54,287 units / gram . I assume this as the activity and declare the result .

Or should I monitor as you suggested  with reference to time ?

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Yggdrasil

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Re: Enzyme Activity
« Reply #4 on: August 11, 2017, 04:57:17 AM »

In general, to measure the activity of an enzyme, you need to setup a reaction with a small amount of enzyme and a large excess of substrate.  You should setup a reaction to monitor the amount of product formed over time (see diagram below), and measure the initial rate of the reaction. 

(image source: https://en.wikipedia.org/wiki/Enzyme_kinetics#Enzyme_assays)

It is important that this initial rate is not dependent on the initial concentration of substrate, so you should setup reactions with varying amounts of substrate and find the point where the initial rate saturates.  If the concentration of substrate is not high enough to saturate the enzyme, your measured rate will not reflect the enzyme's maximum rate of activity.

If you have an excess of enzyme over substrate and are doing an endpoint assay (as it looks like you are doing), the enzyme is likely using up all of the substrate by the end of the reaction, so you are basically just measuring the amount of substrate that you added, not a particularly useful piece of data.  The "rate" you calculate will not reflect the activity of the enzyme because the enzyme will be idle for part of the reaction as it has run out of substrate to use.

Based on the data you provided, if looks like you may want to use at least a 100,000-fold dilution of your enzyme, if not higher.
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ganesh2gig

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Re: Enzyme Activity
« Reply #5 on: August 11, 2017, 06:40:04 PM »

Hey thanks a lot for the information .   

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