Will amylase inhibitors affect the colorigenic reaction between starch and iodine?

Question

I'm doing an experiment for my IB bio EE involving colorimetry. I'm not experienced at all with colorimetry, so I'm having some trouble planning it. The experiment is on enzyme kinetics, and I'm testing the effect of an inhibitor on the rate of digestion of starch by alpha-amylase. Currently, my plan is just to use a starch solution with an iodine indicator, add the amylase and inhibitor and measure the change in absorbance of a certain wavelength over time with the colorimeter, however, I'm worried that when I add the amylase and the inhibitor it'll affect the colour of the solution quite a lot. Will this be an issue, and if so is there a better method I could use to control for it?

Any help would be appreciated, this is my first time actually planning an experiment and I'm finding it difficult :)

Answer 1

Let's work through the options.

First, the amylase could affect the color of the starch-iodine complex by direct association (rather than gradually digesting the starch). I'd think someone would have noticed that by now, but you could test amylase + starch + iodine versus starch + iodine negative control at "time 0" to see what you think.

Next, your inhibitor could affect the color of the starch-iodine complex. In that case, a starch + iodine negative control will look different from an inhibitor + starch + iodine negative control.

Finally, perhaps amylase binds the inhibitor in some special way that causes the whole complex to hunt out starch-iodine complex and make a strange color. It seems unlikely, yet it's not that unlikely since depending how it works the inhibitor might manage to jam up amylase in mid-bite with the starch attached, and the protein somehow manages to mess with the wrapping of starch around iodine. In that case you need to look at whether (amylase + inhibitor) + starch + iodine looks different from amylase + starch + iodine and/or inhibitor + starch + iodine at "time 0", then consider the color change from there.

For "time 0", you might conceivably need to extrapolate back to that time point after measuring changes after some short time intervals you can accurately measure, if the color change is rapid.

Answer 2

To measure your amylase activity, you will monitor the disappearance of amylase’s substrate, starch. Starch reacts with iodine (which is yellow) to form a blue compound (Amax 620 nm). This reaction is the basis of a colorimetric assay for amylase activity. You should be very careful about the volume of the final solutions so it won't effect your results. For example you will add inhibitor to one of them and you won't add that to the other but you should add water instead of the inhibitor so dansity won't be a problem. (final volumes should be same since starch amount at the beginning will be same) You can use some acid like HCL to stop enzyme activity so it won't change the colour and won't effect your results. After the acid stops the enzymatic reaction and the iodine reacts with the starch to produce the blue color. Any starch that has not yet been hydrolyzed by the amylase will turn blue color being proportional to the amount of starch remaining. The intensity of the blue color can be by measured by its absorbance at 620 nm. The greater the change in absorbance between a sample containing the initial amount of starch (without enzyme) and the hydrolyzed mixture containing the enzyme, the greater the amount of starch degraded by the enzyme, therefore the greater the activity of the enzyme being measured. Make sure you give the enzyme enough time and heat to catalyse the reaction. Sorry for grammar errors. I hope this was helpful!

Edit:you asked if inhibitor effects the experiment. I did say it above. If you use a colorless substance to denature or inhibit the enzyme it won't effect your results. If you can't do this you may add the inhibitor after the reaction is over so it won't effect the reaction but you may eliminate possible differences in color