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.
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.
The cuvette that had catechol and the enzyme and was placed in the 60 degree bathshows the greatest absorbance. The higher temperature a cuvette was kept, the more enzymatic activity it displayed through absorbance. Cuvettes containing both catechol and the enzyme solution showed a increase in enzymatic activity as indicated by absorbance.
In the cofactor procedure, the cuvette containing PTU showed the smallest amount of increase between measurements.The cuvette containing EDTA appeared to have inhibited the enzyme reaction, but not as the PTU.
The hypothesis that the cuvette with both the catechol and enzyme solution that was placed in the 37 degree bath would result in the greatest amount of enzymatic activity was not supported, because the cuvette that contained both solutions and was placed in the 60 degree bath showed the greatest amount of enzymatic activity.
The hypothesis that magnesium and calcium are the necessary cofactors for catecholase was not supported, because the cuvette that contained PTU inhibited enzymatic activity much more than EDTA.
Although the hypothesis that the 37 degree bath would result in the most enzymatic activity was not supported, the fact that the cuvettes containing both catechol and the enzyme solution showed a greater increase in enzymatic activity as indicated by absorbance is consistent with the premise that the enzyme acts as a catalyst for the oxidation reaction. We thought that the 60 degree bath would denature the enzyme in the enzyme solution, but it did not.
Human error is an area of weakness in the study. If the parafilm was left on some cuvettes for a different amount of time than others, the data may have been skewed because the reaction depends on the presence of oxygen.
Accuracy is another factor.The absorbance of green light that was measured by the spectrophotometer indicated enzymatic activity, because the benzoquinone reflects orange wavelengths and absorbs green.
The blank cuvettes used in both procedures accounted for the green light that was absorbed by a cuvette with only water, and allowed the calibration of the spectrophotometer so that only the green light that was absorbed as a result of enzymatic activity was measured.
It appears as though the cofactor in catecholase is copper, because PTU binds to copper in catecholase, and the data showed that the PTU prevented the catecholase from catalyzing the catechol in oxidation. (Clapper, A, 2007)
Clapper, A. The effect of temperature and chellating agents on catechol oxidastion. Journal, section 403. March 26, 2007.