For enzyme kinetics, I testes with the enzyme Calf Intestinal Phosphatase, the s
ID: 529596 • Letter: F
Question
For enzyme kinetics, I testes with the enzyme Calf Intestinal Phosphatase, the substrate was p-nitrophenyl phosphate, and the inhibitor was p-nitrophenol. The negative control was the product only ( enzyme + substrate) , the positive controls were the substrate only, enzyme only, and enzyme+inhibitor. Measure absorbance for each control with 30s until 4 min, I got the results below:
What the positive control and negative control meant? how they helped for testing the enzyme and inhibitor?
5 Time (s) Enzyme only Enzyme 0.4mM Inhibitor Substrate only (1.35mM Product only -9.37E-03 -5.95E-04 3.36E-04 30 8.99E-03 4.58E-04 -8.70E-04 2.29E-04 60 -9.16E-04 -1.03E-02 -1.10E-02 -8.85E-04 3.36E-04 -1.10E-02 -7.63E-04 120 2.44E-04 -1.12E-02 -5.65E-04 150 1.68E-04 -1.14E-02 180 -4.27E-04 2.28E-04 210 -1.12E-02 -2.59E-04 3.66E-04 -1.14E-02 240 -3.05E-05 5.19E-04 0.601 0.601 0.601 0.601 0.601 0.601 0.601 0.601 0.601Explanation / Answer
Ans. Aim of the experiment: To assay enzyme activity in presence and absence of inhibitor.
I. Negative control – It is a reaction mixture that lacks a specific component that is responsible for negative result. Here “absence of enzyme inhibition” is the negative result. Therefore, the reaction mixture (enzyme + substrate) lacking the inhibitor serves as negative control because it gives negative result for enzyme inhibition.
II. Positive control: A reaction mixture that gives the desired positive result is called a positive control. Here, the reaction mixture (enzyme + substrate + inhibitor) serves as positive control because it gives positive result for enzyme inhibition.
III. Note: The positive and negative control parameters depend on the “Aim” or desired result of our experiment.
“Substrate only” and “enzyme only” can NOT serve as any control for the given instance where the desired result is “enzyme inhibition” because these reaction conditions, when excluding inhibitor, are NOT sufficient to give any reaction for product formation.
Imagine another scenario where our aim is to “Assay enzyme activity” , but not enzyme inhibition. In this case, (enzyme+ substrate) serves as positive control because it would give the desired positive result (enzyme activity is observed). A reaction mixture containing “enzyme only” or “substrate only” serves as negative control because both of these give negative result (no enzyme activity is observed).
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