I am conducting research about alpha amylase inhibitors. Because of limited reagent, i still use starch-iodine method, but i still don't have a good data of inhibition for determining ic50 (good data means as inhibitors decrease , the inhibition increase or decrease linearly ).
These are some of the things that made me hesitate. 1. I used the drug acarbose (as a comparator to the inhibitor I developed) which I was afraid would destroy the starch as a substrate.
2. The starch-iodine formed was not very stable so when measuring the absorbance some samples faded while other samples were being read so the data was bad.
I used the references as attached
can someone give me some advice?
Your questions are quite puzzling. I am not sure that I really understand what you are asking. I try to answer your questions as good as possible.
1. According to the literature , acarbose has carbohydrate nature and effects non-competitive inhibition of α-glucosidase activity. That is, acarbose binds to amylase and does not interfere with starch (M. Al Kazaz, Eur. J. Biochem. 252 (1998) 100-107).
2. Your starch-iodine absorption values are not stable on storage. Could this be due to interference of iodine with components in your inhibitor extracts? A second explanation is that the inhibition action is a slow process. As a first step I recommend to test the stability of starch-iodine absorbance in the absence of extract.
The second part of my comments aim at putting the starch-iodine assay (SIA) at firmer ground. In the attached Nigerian paper (C. Onomadu et al.) a reducing sugar assay (RSA) is used as a reference method. It must be borne in mind that SIA and RSA depend in a different way on degree of polymerization (P), as shown in the Table attached (Amylase inhibition Septian-2):
The Table shows that RSA has its highest sensitivity at low P (< 10) whereas SIA is sensitive only at P > 10. The results also show that there is no linear relationship of DE, IBC and λmax on P. Because inhibition is more relevant at high P, SIA is preferred over RSA as a tool for monitoring inhibition. The Table comes partly from W. Banks et al., Carbohydr. Res. 17 (1971) 25-33 (attached). This work reports an in-depth study of relationships between iodine binding parameters and amylose P.
Please note that (a) the work of Banks relates to pure amylose and not starch, so by making test samples you have to take into account the amylose content of your starch sample, and (b) λmax is concentration independent.
Thank u for your explanation sir. Actually, in every experiment that i've done, the absorbance of the extract + Starch + iodine is decrease due to the increasing of extract concentration and Sometimes the absorbances is fluctuative so i think the extract influence the stability of a Starch-Iodine. So what do you think? Is there something in your mind that can help me?
I wonder whether you have pretreated the starch. Because of its crystalline nature granular (= ungelatinized) starch is much less susceptible to enzyme action than soluble starch because of its partially crystalline nature. There are two general methods for starch solubilization (gelatinization): (1) dissolution in 1 M NaOH followed by adjustment of pH to the enzyme optimum, (2) cooking in water. Only gelatinized starch is fully accessible to amylase and iodine binding. In your attached paper by Ononamadu et al., no mention is made of any pretreatment. Ungelatinized starch in suspension will settle on standing because of its higher density (ca. 1.5 g/cm3) together with the absorbed iodine. The settling rate is also dependent on the granule size. Slow settling could result in variable absorbance values. Such dependence of absorbance values on standing time (1 or 20 min) is also shown in Table 2 of the Ononamadu paper.
As a first test I would suggest an iodine absorbance assay with only (gelatinized) starch and iodine without amylase and inhibitor. The colour should be stable at least for some time.
A second idea is to do the amylase treatment (with and without inhibitor) separately from the iodine assay. After enzyme inactivation, transfer an aliquot to a separate tube for doing the iodine assay. Colorimetric Iodine assays are very sensitive and require much less starch than enzymatic assays. In this way, possible interference effects of extract components are minimized.
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