The DNS method is used for estimating the concentration of reducing sugars in a sample It was originally invented by G. Miller in 1959. Reducing sugars have the property to reduce many of the reagents. A reducing sugar is one that in a basic solution forms an aldehyde or ketone. The aldehyde group of glucose converts 3,5-dinitrosalicylic acid (DNS) to 3-amino-5-nitrosalicylic acid, which is the reduced form of DNS. Water is used up as a reactant and oxygen gas is released during the reaction. The formation of 3-amino-5-nitrosalicylic acid results in a change in the amount of light absorbed, at wavelength 540 nm. The absorbance measured using a spectrophotometer is directly proportional to the amount of reducing sugar. Aldehyde group , carbonyl group, 3,5 dinitrosalicyclic acid 3amino,5nitro salicyclic acid.
Stock standard sugar sodium: 250 mg of glucose in water and make up the volume to
100 ml.
Working standard sodium: Take 10 mL from this stock solution and make up the volume to 100 ml.
You can make stock glucose at 1mg/ml by adding 0.1g glucose into 100ml distilled water
what about the wavelength 540 nm reading, how i can convert it to the reducing sugar value???
what about the wavelength 540 nm reading, how i can convert it to the reducing sugar value???
what about the wavelength 540 nm reading, how i can convert it to the reducing sugar value???
Dear Aswani Thekkangil, both dextrose and glucose mean absolutely the same. Just mind which form of glucose (dextrose) do you use - monohydrate or anhydrous.
Dear Shahabaldin,
According to the type of enzyme that produce monosaccharide or disaccharide the solution standard would be prepared.If it produces monosaccharide, glucose solution must be used and if disaccharide is the product of the enzyme activity (for example Alpha-amylase) maltose solution must be prepared as a solution standard.
Dear Alaa,
For converting the absorption to the concentration, it is necessary to prepare standard curve which relate the concentration to the absorption.
According to the enzyme and substrate, if you follow the consumption of substrate, it is necessary to
1. Prepare different concentration of substrate.
2. Plot the standard graph of substrate (Adsorption - Concentration).
3. According to the adsorption of enzyme and the standard curve, you can find the concentration of unreacted substrate.
4. From the initial concentration of substrate and unreacted substrate, you can calculate the consumed substrate.
The enzyme activity is calculated according to the following formula:
Enzyme Activity(μmol/min ml) or (U/ml) = (Consumed Substrate) ×Total Reaction Volume / (Reaction time (min)) × (Enzyme volume(ml))
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