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In an enzyme action lab, a student wanted to know the effect of salt concentration on...

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nikigrays | Student | (Level 1) Honors

Posted April 12, 2013 at 11:33 PM via web

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In an enzyme action lab, a student wanted to know the effect of salt concentration on the action of an enzyme. Describe how you would set up the positive and negative controls for this experiment.

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Wilson2014 | eNotes Employee

Posted May 11, 2013 at 12:01 AM (Answer #1)

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In an enzyme lab, one would generally perform an enzyme assay with varying conditions to determine how the rate of the enzyme is affect.

If we are testing the effect of salt concentration, then the concentrations of the enzyme itself and its substrate must remain constant in the solution (and obviously the temperature and other external factors that might influence your data).

Note: The concentration of the enzyme is very important because if the concentration is too high, the enzyme assay would produce an initial concentration that is too slow because once the enzymes are injected into the solution with the substrates, they will begin converting the substrates into the final product; however, as more products are being produced, the equilibrium will be shifted such that the enzyme will start converting the products into the substrates. So a high concentration of enzymes quickly produces a high concentration of products before you will have the time to your solution into the spectrophotometer for the enzyme assay. Since not all of the enzyme activity is dedicated to a forwards reaction, the enzyme assay would not be accurate. We want to dilute the enzyme concentration such that the concentration of the substrate is highly greater than that of the product when we have situated the solution inside the spectrophotomer so that all of the enzyme activity is dedicated towards converting the substrate to product and this is what you are measuring to determine the rate of enzyme activity.

A positive control for this type of experiment would be to perform an enzyme assay without addition of any salt. Just measure the rate of enzyme activity in its standard condition (with just the substrates it binds with and catalyze). This control allows for you to compare and observe how the addition of salt influences the rate of enzyme activity from its standard condition.

A negative control would be to have every chemical compound in the solution, including the salt, but leave out the enzyme. The purpose of this control is to ensure that nothing else is affecting the spectrophotometer's reading of your enzyme assay other than the enzyme itself. If your assay tells you that there is a certain enzymatic rate, then that means your solution is not purified of other enzymes that also uses up one of your substrates or that there is a significant external factor, such as oxidation of your substrates, which are affecting your experiment. Usually, if all materials/compounds/biomolecules are 100% pure and you perform the procedures correctly, this control is less useful than the positive control. 

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