Normally it is said that dns is added to stop the reaction in enzyme assay, what happens to the enzyme.
DNS binds only to reducing sugars (eg. glucose) and cannot bind to non reducing sugars (eg.sucrose).During the incubation period of this experiment, enzyme acts upon sustrates to give products which are reducing sugars.Then you add DNS.Then, the next step is heating the reaction mixture which is responsible for 2 steps, one for heat inactivation of enzyme and other for efficient binding of reducing sugars to DNS to give ANS which shows maximum absorbance at 540 nm.
Generally, the enzyme substrate reaction is stopped by heat inactivation, or by changing the pH of the environment, or by the use of specific inhibitors. DNS is mainly used in detecting/ quantifying the alpha amylase activity. Reducing sugars produced by alpha amylase reacts with DNS and produce ANS which absorb the light at 540nm. DNS reacts with reducing molecules.
If I may add, I don't know if DNS actually inhibits an enzyme catalyzed reaction due to its presence (e.g. by competitive or non-competitive inhibition). The protocol for developing colour in the DNS assay for reducing sugars is to heat the reaction cocktail in boiling water for approximately 5 minutes. This step is likely to stop enzyme catalyzed activity due to heat-induced denaturation of the enzyme. I hope this helps.
DNS binds only to reducing sugars (eg. glucose) and cannot bind to non reducing sugars (eg.sucrose).During the incubation period of this experiment, enzyme acts upon sustrates to give products which are reducing sugars.Then you add DNS.Then, the next step is heating the reaction mixture which is responsible for 2 steps, one for heat inactivation of enzyme and other for efficient binding of reducing sugars to DNS to give ANS which shows maximum absorbance at 540 nm.
DNS reagent has an approximate pH of 12.5-12.6. Addition of 3ml of DNS reagent to a simulated reaction mixture of 1 ml of 0.1N citrate phosphate solution and 1ml of water (in place of enzyme and substrate) gives the solution a pH of approximately 12.3-12.4. As Elavarasen said, the reaction can be stopped by altering the pH environment. This elevation in pH should be sufficient to stop the reaction depending on your enzyme. Stopping the reaction by the addition of DNS and immediate immersion into an ice slurry should stop the reaction.
sorry for the year late answer but this just came up in a Google search I was doing.
Dear all experts.... M checking the alpha amylase activity on starch by DNS method with buffer 7.0, 8.5, 9.5 and 10.5 and temperature ranging from 15 degree to 55 degree.... Can u please suggest the protocol....
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