I have followed the procedure that you provided to determine the activity of the beta glucosidase, however what I am unsure of is how the rate is calculated?
In order for me to establish the rate, I followed your instructions however incubated the enzyme/buffer and PNPG solution for different times, ranging from 0-16 minutes (in steps of 2 minutes) and constructed a calibration graph - which I have attached.
I also constructed a 4-Nitrophenl calibration curve, also attached in which the concentration ranged from 0 39 nmoles.
The beta Glucosidase which I used has an activity of 7.55 Units/mg. It releases 1 micromole of product per ml.
Following the instructions from the assay provided by you;
I made my initial enzyme solution at 1 mg/ml. To then ensure that this was 0.006 Units/ ml i added 0.007947 mls of this to 9.992 mls of the 0.2% BSA solution.
I then followed all of the instructions provided, but varied the incubation period to obtain the calibration - can you advise if this was correct?
The instructions state that the enzyme solution be incubated for 15 minutes, I am therefore assuming that the absorbance reading, once superimposed on the 4-nitrophenol calibration would represent the amount of product released after 15 minutes? This is where I am getting confused.
When I calculate the rate, I take the x value from the beta glucosidase assay and replace y of the 4nitrophenol calibration equation with it:
4NP: y=0.0153x + 0.1215
BG: y =0.0851x
x = 2.38 n moles
with 0.079mg of protein present (is this correct?) I would expect 0.079 micro mole of product to be released per minute, however only 0.0023 micro moles were?
Can you advise if i have calculate correct,and where i am going wrong please?
Your calculations and dilutions to produce the 0.006 U/ml enzyme solution are correct.
However, you should NEVER incubate the solutions for different times. This is your fundamental mistake. Do all incubations at 15 minutes just like the procedure instructed.
But what you do differently is have different amount of p-nitrophenol in the tubes (for the construction of the calibration curves). You don't need to construct a "curve" for the tubes with the PNPG. What you do is construct the calibation curve with the p-nitrophenol and then use that curve to determine the production of p-nitrophenol in your one or two test solutions. If you do it right, you only need to do one test solution. Therefore, your first graph in the attachment ...
This solution shows how to calculate the rate of Beta Glucosidase Activity