I'm new to this and need a little bit of help. How do I quantify the "total amylolytic activity" of a mixture of amylolytic enzymes using the DNS method? I read from the attached file that they measure the amylolytic activity by this method. Thanks!
hi, this method have name Bernfeld methods :)
Bernfeld, P. (1955) Amylases, alpha and beta. Methods in enzymology I: 149-158.
1. Prepare solution of glucose in dist water (water, 0.1 mM, 0,5 mM, 1 mM, 2,5 mM, 5 mM, 10 mM).
2. Prepare ans set your spectrofotometer at 550 nm.
3. Prepare DNS.
4. Prepare boiling water bath.
5. Prepare solution of a-amylase.
6. Make zero in spectrofotometer on a water (or mix of water, DNS and enzyme).
7. Add 1 ml DNS, 1 ml of each calibration solution glucose + 5 ml water dist. Put in water bath 100 C at 5 minute.
8. Measure absorb. Notes response from spectrofotometer for each point. Should be 6 point of calibrat curve.
9. Build calibartion curve. (Think which type of zero sample you need water? or mixture water, DNS and enzyme?
10. and make yours measurment with biological sample in triplicate and done :)
!!!!!This method is general - cant be used for quantify of glucose!!!!!!
Dear Yong,
please forgive me but this subject is from 1955 years.... (basic of sugar analysis)
Word copy of the original DNS method for reducing sugars .. good luck
Dear Mariusz, why DNS can't be used to quantify glucose!?
Thanks all.. Mariusz, precisely because Bernfield's paper is an old paper that's why I want to keep a copy of it in my archive! It's always good to get the basics strong, more so for me as a new comer in the field.
Hai young,
prepare sugar solution ( as u desire) in a concentration of 10 - 50 micro gram as a standard. made up to 2ml with distilled water , and add 2 ml of DNS SOLUTION in all , take reading at 570 nm.
Dear Ahmed Mohy, how you see problem is in amylolytic enzymes not water and glucose. Method with spectrofotometer is old and require pure analytes. In fact, amylolytic enzymes give NOT ONLY GLUCOSE give my h more sugars (forgive me but this is subject from books for students). I think we don't have time for explanation this old problems :) . I propose for quantify hplc with RI in Rezex column or gc-fid after derivative with HDMS in piridine on HP-1 or HP-5. I can send some chromatograms from that kind of analysis.
To Ahmed Mohy
When amylases hydrolyses the starch , the solution is a mixture of oligosaccharides and maltose, glucose , all reducing sugars. Then not only glucose will be evaluated by DNS but the other reducing molecules .
Glucose can be determined by glucose oxidase or by HPLC
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