i have prepared glucose standard graph and protein standard graphs and have obtained the mg/ml of glucose and BSA for 1 OD..
simultaniously i have done the enzyme assay and protein estimation.now i need to find out the enzyme activity and specific activity..help me derive the formula for the protocol i have used in the attachment for both.
please help
Hi there,
First thing you have to make sure of is that you are actually measuring the initial velocity of the reaction (if not the activity of the enzyme will be underestimated). How to do that? Just by following the kinetics. In the assay described incubation is 1hour. In the same condition, consider time points between 0 and 1 hour (15, 30, 45 and 60 minutes for instance) and run the assay. If initial velocity condition applies you should observe strict linearity between time and product (ie velocity of reaction is constant). If not you will have to shorten incubation so that linearity is respected.
If OK for initial velocity then the calculation for enzyme activity is :
Glucose determination is made from 50µL of the assay that you mix with DNS in 2mL final volume. x is the actual glucose concentration in mg/mL for this mix, right? So the actual concentration of glucose in the assay is 40 times higher as 50µL is 1/40th of 2mL, OK? This concentration of glucose has been obtained from 1h incubation with 100µL of enzyme solution therefore enzyme activity is (40x)*10 expressed in mg of glucose formed per hour per mL of enzyme. For the specific activity , you only have to divide the activity by enzyme solution protein concentration expressed in mg/mL (taking into account that protein determination is made from 50µL protein sample in 3.25mL final volume) . You will obtain AS in mg of glucose per hour per mg of protein.
Using Standard curve
Activity =___ moles of the product(glucose) released/ min/ mLof enzyme source
Specific Activity = Activity / __mg of protein= _____moles of glucose / min/ mg of protein
Hello Dominique
Thank you so much for giving the detailed solution to my problem......i stiil have some doubt , that after taking 50µL and adding it into 2ml DNS. i am boiling it for 10 min and then diluting it further by adding 1.25ml of water.so will this dilution be included to find out the enzyme activity or this will not be included.
if yes then what will be the enzyme activity ?
Hi again,
OD measurement is made on the final solution so dilution has to be taken into account. I didn't pay attention to this 1.25mL. So dilution factor of 65 (50/3250) should be used instead of 40 in activity calculation.
I am using absorbances to calculate specific activity of Ache. The formula is A/el * 3 * initial volume/0.1 * dilution factor.
I have used dilutions of 1 in 3.
I am confused to what I should write the dilution factor as. Is it 3 or 1/3.
Any help will be great.
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