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(Solved) (Latest ver. Aug 2020) - Beta Glucosidase Activity

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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;
(http://www.calzyme.com/commerce/catalog/spcategory.jsp?category_id=1026)

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 is 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

therefore 0.0851=0.0153x+0.1215
x = 2.38 n moles

with 0.079mg of protein present (is this correct?) I would expect 0.079 micro moles of product to be released per minute, however, only 0.0023 micro moles were?

Can you advise if I have calculated correct, and where I am going wrong, please?

 







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