I am currently exploring the ability of my enzyme to act on different sugars as part of my project to alter the substrate specificity. To measure the enzyme kinetics I use a coupled assay whereby the hydrogen peroxide produced in the catalysed reaction is used by horseradish peroxidase (HRP) to oxidise ABTS resulting in a green product which can be detected at 415nm. The assay works fine with most of the suagr substrates I've tested (galactose, glycerol, xylose, sucrose, maltose...) but with fructose, arabinose and mannose I get formation of the green colour when I just add the ABTS and HRP to the sodium phosphate buffer containing the sugar ie. before enzyme is added. This green colour fades after a few minutes and seems to be dependent on the concentration of the sugar (higher concentration result in a more intense green). If I wait for the colour to fade and then carry out the assay, activity is much lower than it should be suggesting that one of the components is depleted or the conditions in the cuvette have changed.
I've tried using ABTS and HRP from different sources with no change. The fructose is from a 10 year old tub which has worked fine for me in the past and the mannose and arabinose are less than 6 months old, from two different suppliers.
Please let me know if you have any idea what could be going on and what I could try to fix it!
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