Can anyone interpret why the absorbance (DNS assay method) in alpha amylase assay get increased with increased standard (inhibitor) concentration? it should be decreased with increased standard or sample concentration
https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/enzyme-activity-assays/enzymatic-assay-of-a-amylase
The absorbance in the DNS assay should increase in direct proportion to the product or standard (maltose) concentration, within the linear range of detection.
If the inhibitor alone (no enzyme reaction) is giving a signal that increases in direct proportion to the inhibitor concentration, it means either that the inhibitor reacts with DNS, or it is contaminated with something that reacts with DNS. If this signal is relatively small compared to the signal from the enzyme reaction, you can measure and subtract it from the complete reaction signal. If the signal from the inhibitor alone is comparable to or larger than the signal from the complete reaction, you will have to purify the inhibitor, or find a better source of it, or use something else.
Thanks for the answer
I am measuring the inhibitory activity of some samples against alpha amylase using Acarbose as standard drug. Both the std. and samples have the same pattern (absorbance increases with increased inhibitor concentration). I tested DNS with serial dilutions of amylase and it works nicely, however in the main experiment I got these results.
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