My positive control (conc: 4, 8, 12, 16, and 20 ug/mL); acarbose had an unexpected result, my least concentration for acarbose is 4 ug/mL and it actually showed the highest %inhibition for alpha amylase. and the trend is actually falling down as the conc increases from 4 to 12 ug/mL and the trend is rising again from 16 to 20 ug/mL
I used 0.5 mg/mL enzyme solution in 0.02 M sodium phosphate buffer, containing 6mM NaCl. 1% starch.
Can someone help me why is this happening? Thank you.
Thank you Dr. Tranchant, I also obtained the IC50 using graphpad prism and it showed ic50 hill slope of 0.3. I have also read about in vitro biochemical assay guide that if an IC50 of an inhibitor has a hill slope of < 1, it is a sign of an enzymatic contamination. What can you say about this? Thank you.
Thank you very much sir! Your help is very much appreciated.
Sir, I also have a question regarding the IC50. Is it possible to obtain IC50 value without getting a 50% inhibition in any of the concentration? Thank you.
Ok sir. Lastly, regarding the possible hydrolysis or isomerism of acarbose as described by Kim et al. (1999) they used a-amylase from Bacillus stearothermophilus. Is it applicable to my scenario wherein i used a-amylase from pig pancreas (bought from sigma-aldrich now merck)?
thank you very much sir, your help is greatly appreciated!
Ohhh i see. So contamination is one possible cause i think?
Hi Romil Steve Pongcol Mazo, we have experienced the same issue at our lab. As you mentioned, in our first assays, the lowest acarbose concentrations resulted in the lowest absorbance measurements, giving as result the highest inhibition values. Thus, as the acarbose concentration increased, inhibition was the lowest. We realized that we were actually measuring the acarbose concentration as it reacted with DNS (acarbose is a maltotetrose analog, possessing a free end that may act as a reducing agent). Perhaps, if you re-analyze your data you will find a nice positive linear correlation between the acarbose concentration and the absorbance. So, Why was acarbose reacting with DNS rather than inhibiting alpha-amylase?
Well, it resulted that our alpha-amylase solution was old (it was prepared one week before starting the experiments) and thus we think that the enzyme suffered an important degradation and was no longer capable of interacting with acarbose. The same experiments, performed with a freshly prepared enzyme solution, resulted in the expected inhibition behavior. Furthermore, we observed that after the third replicate in a single day, absorbance values started to decrease and a new enzyme solution was required.
Hope this will help with your problem. Good luck!
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