Enzyme Calibration Lab

Introduction
Enzymes are proteins that increase the speed of reactions in cells. They are catalysts in these reactions which means that they increase the speed of the reaction without being consumed or changed during the reactions. Cofactors are required by some enzymes to be able to carry out their reactions by obtaining the correct shape to bind to the other molecules of the reaction. Chelating agents are compounds that can disrupt enzyme reactions by binding to metallic ions and change the shape of an enzyme. Catechol is an organic molecule present under the surface of plants. When plants are injured, catechol is exposed to oxygen and benzoquinone is released because of the oxidation of catechol. Catecholase aids in the reaction to produce

A low absorbency would have a low color change so would be clear or slightly clear by the end of the trails and a high absorbency would have a strong red color by the end of the experiment.

Methods and Materials This experiment requires four tubes with an enzyme solution, chelating agent and deionized water. Also a fifth tube that is the calibration tube for the spectrophotometer, which only has 5ml of dH2O. The calibration tube is used to level out the spectrophotometer to zero before each trial. The spectrophotometer was set at 540 nm, “since green is not a color seen with the conversion of catechol to benzoquinone.” The enzyme solution was made by using potato that was peeled so that the golden color of the skin wouldn’t react or interfere with the red color needed in the spectrophotometer. After it was peeled, it was cut into chunks to minimize excess heat created while it was blended. It was put in a chilled blender and 500ml of deionized water was added. Chilled, deionized water was used because it created a hypotonic environment that caused the cells from the potato to burst and release the catecholase. It was chilled

EDTA, the chelating agent that binds with magnesium, had a high absorbency and strong color change to red. The correct cofactor was copper which with the chelating agent of PTU and citric acid which both bind strongly to copper which keeps it from binding with the enzyme. This was determined because in the trails, both PTU and citric acid had low absorbency and were clear or roughly clear in color. The catechol in each tube, which was the control for this experiment, allowed the cofactor that would be used in this reaction to be singled out. The way each chelating agent would affect the different cofactors displayed which was not needed for the reaction and which cofactors were needed for the reaction. An inconsistency that may have affected the data would be if the calibration tube malfunctioned in balancing the spectrophotometer to zero. There also could be errors if the calibration tube wasn’t used before each tube was tested in the spectrophotometer. The relationship of the cofactor and amount of enzyme activity would be that if the cofactor is inhibited or not, the enzyme activity would be higher if the cofactor is not inhibited but lower if it was inhibited by the chelating