Kindly send some calculations for the given assay.
I want to follow the given protocol.
In vitro inhibition of α-amylase
The α-Amylase (0.5 mg/ml) was premixed with extract at various concentrations (100-500μg/ml) and starch as a substrate was added as a 0.5% starch solution to start the reaction. The reaction was carried out at 37°C for 5 min and terminated by addition of 2 ml of DNS (3,5-dinitrosalicylic acid) reagent. The reaction mixture was heated for 15 min at 100°C and diluted with 10 ml of distilledwater in an ice bath. α-amylase activity was determined by measuring spectrum at 540 nm. The % α-amylase inhibitory activity is calculated by the following formula:% Inhibition = (Control OD-Sample OD / Control OD) × 100
There should be no problem with preparing suitable standards. The DNS method measures reducing power which in the case of amylase acting on starch will mainly arises fro maltose and some maltotriose. Assume that all the reducing sugar is maltose, so prepare a solution of maltose of known concentration and take samples covering a range of volumes and add water to each to ensure that the combined volume of water and standard is the same in each case. You will know the concentration of maltose in each of the samples.Treat the samples in exactly the same way as the samples from the enzyme assay. Make sure that the total volume of the mixture used in the DNS assay is the same for each standard and enzyme mixture. Make a plot of Absorbance against maltose concentration and from this determine the maltose concentration in the samples taken from the enzyme assay.
This is basic information that you should have known already before attempting any analytical or enzyme work
Make a standard curve using maltose. Choose these concentrations based on the detection limits of your instrument and expected values in your assay. Readings from your actual assay should be in the range of standard curve generated. Ideally, the standard curve should be part of your assay with the same total volume of each reaction mixture.
And this applies to any other assay and is part of basic lab courses.
My favorite starting point for enzyme projects is the BRENDA database. This is the page listing over 1200 substrates for alpha amylase, including colorimetric substrates, and links to the relevant journal articles that describe the assays.
https://www.brenda-enzymes.org/enzyme.php?ecno=3.2.1.1#SUBSTRATE
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