We set up a standardcurve with standards from 0, 8,20,40,80,200,400 and 800 mU of the endproduct 4- methylumbelliferone in 200µl. Together with an enzyme reaction (sample) which contains the same buffer and in all reaction the same amount of MUG (200mM). The only differs from the amount of beta-glucuronidase.
The enzyme reactions contain 0, 0.5, 1, 2, 3, 4 and 5 µl beta-glucuronidase.
We do a kinetic measurement of 15 minutes with an interval of 1 minute. The excitation wavelength is 360nm and the emission is 535nm. The measurments are done by an Infinite 200PRO.
We get a standardcurve from the standards where mU are the X value and the RFU are the Y. How can we calculate the activity of beta-glucuronidase (mU/µl) in the enzyme reactions?
You start by measuring the initial rate (slope) of the reaction progress curves, in RFU/min. You then convert RFU to mU (I'm not sure what mU is, by the way) using your standard curve, giving you mU/min. Divide by the number of µl if enzyme to get mU/min/µl. That is a measure of specific activity of the enzyme. If you know the protein concentration in mg/ml (=µg/µl), you can convert to mU/min/mg.
I believe you take the slope of the initial rate RFU/min and multiply by 15 min to get the total RFU. With your standard curve, you can then calculate the mU from the RFU. Then you divide by the ul of enzyme used. Or the ul of the reaction volume. I am confused by what you mean by ul. So you will have to clarify.
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