Enzyme Lab Report
An enzyme is a protein molecule that speeds up the rates of chemical reactions by many folds. They recognize, bind, and change specific reactants. They do not change thus can catalyze the same reaction again and again. Activation energy also known as an energy barrier is the amount of energy needed in order to begin a chemical reaction. Catecholase catalyzes the reaction rate of catechol oxidation. Catechol is found beneath the skin of many plants such as apples and potatoes.
When it is exposed to air, the oxygen in the atmosphere oxidizes it to benzoquinone, which acts as an antiseptic for the plant. When produce is stored in a freezer, it will stay for long without changing color. This is due to the cooler temperature preventing the catechol in the produce from oxidizing as quickly as it would at room temperature. (Clapper, A, 2007) When cut open, potatoes turn to a brownish color which indicates enzymatic activity. Ethylenediamine tetraacetic acid (EDTA) is a preservative.
The magnesium and calcium that EDTA binds are the cofactors used by the enzymes of bacteria and fungi that can spoil food. Benzoquinone reflects light of orange wavelengths and absorbs light of green wavelengths, which makes us measure enzymatic activity by measuring light absorbance. There is a hypothesis that enzyme kept at 37 degrees Celsius will show the most absorbance which shows most enzymatic activity. Also calcium and magnesium are hypothesized as the cofactors necessary in the functioning of enzymes in bacteria and fungi that spoil food, due to EDTA binding to them. (Clapper, A, 2007) Material and Method Spectrophotometer cuvettes, A spectrophotometer, 18 mL of catechol solution, 10 mL of enzyme solution, 531 mL of distilled, 2 mL of EDTA and 2 mL of phenyl thiourea (PTU) were used as possible chelating agents, 5 labeled pipettes, around 14 pieces of parafilm, kimwipes, cuvette rack, 37 and 60 degree Celsius water baths, one uncooked potato, potato peeler, refrigerated blender, 500 mL of the cold distilled water, and several squares of cheese cloth were used in this experiment.
Method. In preparing the catecholase extract, a potato was skinned, washed, and then diced. Water was added to the diced potato and blended for two minutes. The solution formed was filtred and the extract stored in a container. Eight individually labeled spectrophotometer cuvettes were prepared using different amounts of the following reagents: a buffer of pH 7, a 0. 1% catechol substrate, and distilled water. The wavelength of the Spectronic 20 spectrophotometer was set at 540 nm.
To calibrate the spectrophotometer at zero absorbance, blank control cuvettes prepared with no catechol substrate and labeled “tube 1” was inverted and inserted into the spectrophotometer. The extract to be tested was added immediately to each cuvettes before they were placed to the spectrophotometer and each cuvettes was inverted and placed in the spectrophotometer. The absorbance was read for time zero (t0), the ten minute mark (t10), and each minute in between and recorded.
The cuvettes received the enzyme solution last to prevent the reaction from increasing until we were ready to begin measuring the reaction. The cuvettes were shaken frequently to ensure that the contents were properly mixed. After ten minutes, the cuvettes were removed from their environments. To conduct the cofactors procedure, the cuvettes were prepared first. Cuvettes 1, 2, and 3 received all contents other than catechol, at first. Cuvette 1 received 1mL of enzyme, 2mL of catechol, and 2mL of EDTA. Cuvette 2 received 1mL enzyme, 2mL catechol, and 2mL of PTU.
Cuvette 3 received 1mL of enzyme, 2mL of catechol, and 2mL of dH2O. Cuvette 4 received 5mL of dH2O. The enzyme solution and chelating agent were allowed to sit for a minimum of 10 minutes before the catechol was added, so as to ensure mixing. The cuvettes were inverted and shaken every 2 minutes to continue mixing. After ten minutes of mixing, the catechol solution was added and initial measurements were taken like in the temperature procedure. Next, the cuvettes were placed into a 37 degree water bath and ten minutes elapsed before a second set of data was recorded.
Two data charts were created to record the data for the temperature and cofactor results. The charts show the absorbance rate for each cuvette for the initial readings and the readings after ten minutes. The blank cuvettes, in each procedure allow us to calibrate the spectrophotometer. When the spectrophotometer is calibrated with the blanks, we are subtracting the amount of absorbance that occurs when only H2O is present in the cuvette. This allows us to measure the amount of enzymatic activity indicated by absorbance as we control absorbance that is not due to enzymatic activity.