Currently I am working with alpha amylase assay using DNS method but I have a problem with the colour which is not changing even I used high concentration from the phenolic standards (Trolox and Gallic acid) and I don't know what is the problem.
I used different concentrations from enzyme (0.1 - 1 U/ml) with soluble starch 1% (w/v) but the colour of samples very close from the control (orange-red colour). In addition, the absorbance of standards or samples are higher than the control, and the absorbance increases with increasing the concentration of standard whereas it should decrease with increase the concentration of standard or sample. As a blank, I tested starch with DNS, the enzyme with DNS and different concentrations of standard with starch and DNS, but I didn't get changes in the colour.
The protocol that I used is:
100µL of buffer or sample + 100 µL enzyme (procine pancreatic alpha amylase) then mixture incubated for 10 min at 25°C, add 100 µL of starch 1%, then mixture incubated for 10 min at 25°C, after that 200 µL of DNS (1%) was add to terminate the reaction and the mixture was incubated for 5 min at 100 °C, cooled to room temperature and then solution was diluted by 3 ml of H2O. The absorbance was recorded at 540 nm.
Can anyone tell me what the problem is or suggest what I can do to solve this problem.
Thanks in advance
Hi Mahmoud,
I am not exactly sure about this specific method. So, I have some questions for clarity. You are using soluble starch and adding different concentrations of alpha amylase to it to hydrolyze the starch into oligosaccharides, correct? When this occurs, the absorbance at once concentration of enzyme will increase because DNS binds to reducing sugars (which increases as you hydrolyze the starch). However, if you compare 0.1U to 1 U, than the 1U will create more dark red colors than the 0.1U as it is stronger. Are you trying to measure enzyme activity? Or are you trying to quantify the amount of reducing sugars in a sample? Does any of solutions contain NaOH? When I did this assay, I remember using NaOH (either mixed with DNS or somewhere else) and then heating it. This resulted in various shades of blood red to peach. Hope I can help.
Marsha
Thank you Marsha for your replay, actually I am trying to calculate IC50 or EC50 from my samples (plant extracts) to see if it can use as inhibitor for this enzyme (antidiabetic inhibitor). For DNS reagent contains NaOH (2 M). The problem that the absorbance of standard or my samples are increasing with increasing the concentration and this is wrong. I will appreciate you help to solve this problem or to know what I can do?
Thanks for your interest
Absorbance is increasing with increasing concentrations of your inhibitor?
Also, depending on the temperature-activity relationship, the alpha amylase may not be active at 25C but is somewhere
Some more thoughts:
Try boiling the DNS solution longer than 5 min. Even though the water bath is at 100C, the internal solution temperature may not be and this affects the reaction kinetics needed for the DNS to covalently bind to the reducing sugars. The longer you boil the deeper the color should be.
Secondly, try instead of 1% starch, use 1% glucose or fructose and try the method (without enzyme) to see if the DNS solution is working too. Also, the color should increase with increasing boiling time.
Thirdly, are you using potato starch? Corn starch? I ask because you indicated that it was 1% w/v and I am not sure if it was solubilized before the assay. Not to insult you or anything, just really trying to help. I often have to go back and think about every aspect of the assay to make sure it is logical when I do mine. Anyways, for either starch form, you must boil it first to get it in solution so that the amylase can attack it. I have a paper on the susceptiblity of amylase and glucoamylase to the different starch forms and soluble>swollen/gelatinized>insoluble/granular is the order it works. Potato can go in solution in about 3-5 mins of continuous boiling and corn starch needs a bit more help (but it will become gelatinzed/swollen, but it will less likely be reproducible). This could be a simple mistake.
If none of that works, I would try simply using an glucoamylase since alpha amylases do not readily make reducing sugars like glucoamylases and the assay would probably be more effective if the enzyme swap was performed. Alpha amylase will make some oligosaccharides and the increase in color will not be as obvious if you produced monosaccharides. It is similar to having a necklace and if you have more broken pieces you have more binding ability for DNS. This is why when you measured the starch + DNS you got very little to no color. Hopefully, if you measure the glucose, you should get a ton of color.
Lastly, I found this method from a dissertation online and maybe it would work better since it seems to be similar to what your are trying to do. They use arcabose as a positive control and they use a nitrophenyl-glucopyranoside solution to develop the color. This method does not require the external heating (which may make the results more reproducible as well). See pages 13 and 19.
Anyways, sorry so many messages. I understand how assays can be frustrating and it took me a little while to fully understand your challenge. I hope this helps and that you are able to measure the IC/EC of your inhibitors.
Marsha
http://digitalcommons.library.umaine.edu/cgi/viewcontent.cgi?article=1167&context=honors
I agree with all answers, try to raise the temperature reaction and be sure the pH buffer is óptimal for your amylase. In addition, the amylases require Ca2+ as cofactor, you need to add to reaction buffer.
Thanks for all of you, I increased the temperature to 37 °C and decrease the volume of enzyme against the substrate and sample volumes but what I could not understand that how can the absorbance and reaction colour increased with increasing the sample concentration which meaning lower inhibition with increase the sample concentration!!! Is anyone have an explanation for that???
I mean that when I increased the sample concentration I got darkness "red-orange colour" and higher absorbance more than the control (H2O+enzyme+substrate+DNS).
Thanks in advance.
Dear just put 4-5 ml deionized water in entire assay before taken OD (optical Density) I hope it will solve your problem.
Mahmoud Hefny Gad Marsha Cole Hi, I want to know did you solve your problem and how did you solve? Recently I did quite similar experiment like what you did before, I use precooked bean peptides (simulate gastrointestinal tract) and did alpha-amylase assay, but I met same problem with you. I don't know how to do now, can you help me to solve these problems?
this article is my protocol. the experiment method is quite similar as yours.
hi, i am not sure but did you check the amount of sugars present in your sample. if your sample has free sugars then it might be a problem .
Kindly provide the procedure for standard curve plotting taking maltose as standard. Should I follow the same procedure adding enzyme, substrate and DNS reagent or adding only enzyme or substrate or DNS solution ?
Zhujun Qiu @ Mahmoud Hefny Gad Mina Karimi-Avargani Sadia Zulfiqar
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