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 .