I am doing an assay for Carbonic anhydrase, with Sodium Bicarbonate as substrate. I find that the non-enzymatic rate itself is high, and hence am not able to detect a much steeper activity with the enzyme, before the reaction plateaus. Could anyone suggest a way to reduce the non-enzymatic rate?
Did you mean reaction in the blank controls was observed without enzyme? you may reduce the concentration of the substrate; increase the enzyme concentration to make the difference more significant from the control; or replace substrate with a lower background one.
Normally, as long as the assays are robust from experiment to experiment, using the reaction rate subtracted by blank control's would be better than using endpoint reading and should generate satisfactory results, even if there is background in blank controls.
Actually the reaction catalysed by carbonic anhydrase is extremely rapid. So if I reduce the substrate or increase the enzyme, the reaction will get over even before I place the cuvettes in the spectrophotometer.
I use time course measurement now. End points differ from reaction to reaction. The pattern is reproducible, but not the numbers. I guess this is mainly because the substrate stock solution I use itself starts dehydrating over time.
Are you detecting CO2 formation? Is the pH suitable for this substrate? You may consider contamination as well in the blank sample if the reaction is extremely fast.
Run assays at lower temperature normally helps for fast reaction.
I am detecting formation of hydroxide ions, using a pH indicator and buffer system (khalifah et.al.). I monitor the increase in absorbance of the indicator, as pH increases. I keep the reaction components cold, before adding them. And I do not think it is contamination, as I have repeated it may times.
Using buffer of pH 6.8, non enzymatic rate is very high. If i increase pH (7.2, 7.6), non-enzymatic rate becomes negligible. But the enzymatic rate vanishes as well.
If you have any enzyme specific inhibitor, and see if the non-enzymatic rate in blank can be inhibited then you will know if there is any contamination, which can be from reagents or from labware as well.
Good luck
There are enzyme specific inhibitors. The only problem for which I need a solution, is the non-enzymatic rate. If that is inhibited, the problem is almost solved. But I do not know how to inhibit Bicarbonate from decomposing.
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