Hi everyone,
I am wondering how one would determine precisely what is the optimum pH or temperature of the alpha amylase in a enzymatic assay?
My problem is that I am using 4 different enzymatic reactions to get a colored product that I can quantify with the spectrophotometer.... Then, if one changes the pH, one would affect all enzymes in this assay not only the alpha amylase...
Thanks for your help,
Fab
Thanks Peter.
I agree. But I'm trying to show students how to determine the optimum pH of α-amylase in an enzymatic assay. And the only way I found to measure its activity is by using these 4 different enzymatic reactions that form quinoneimine. I'm wondering how do we know that optimum pH of salivary α-amylase is exactly 5.6?
Maybe I should change with an enzyme that can directly make a colored product.
Since this is a student exercise, switch to a simpler detection system or use an enzyme that has a simpler method of detection. For example, you could use glucose 6-phosphate dehydrogenase and follow NADPH formation at 340 nm.
If this is not possible and you wish to continue with your current system, you need to set it up so that the coupling enzymes are present in such excess that a partial loss of activity due to pH change would not matter. To test for this condition, perform your complete reaction at each pH at a single concentration of amylase (the highest concentration you expect to use) while varying the concentration of the coupling enzyme system (keep things simple by using the coupling enzymes in a fixed ratio). Identify a concentration of coupling enzymes that is more than sufficient to achieve the maximal rate of colored product formation at every test pH.
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