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In order for the 4-nitrophenol to release the yellow colour expected, it has to be fully ionised. With a pKa of 7, I have decided that pH9 would allow it to be fully ionised - this is also supported by the yellow colour given off, and a reasonable calibration curve being constructed.

My problem is this:

The beta-glucosidase that I am working with is in a 10mM citrate buffer at pH 5.2, not pH 9 as required. When the substrate, p-Nitrophenyl β-D-glucopyranoside is added to the enzyme, a yellow colour is released, however it is not as intense as the colour given off from the 4-nitrophenol at pH 9. I then deduced that if I carried out the reaction at different time frames (ie buffer, enzyme and substrate for 1minute) and added a known quantity of sodium carbonate to ultimately stop the reaction and bring it to pH 9, I would get a range of results (timeframe would be 1-5 minutes each giving different absorbance readings) which would form a straight line, and thus allow the rate of reation to be determined. Sadly this does not happen and all absorbance readings are the same.

Can anyone advise of either an assay which I can carry out at pH5.2 fr beta-glucosidase, or another way to potentially overcome this problem.

It may worth noting that there is a chemical shift in absorbance depending what pH the readings are taken at.

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