Enzymes are the the reason why our world goes around, actually they are the reason why we have a world that goes around. Enzymes are the reasons why multiple reactions occur in our world 24/7 because they allow the cell to carry out the chemical reactions. These reactions are why cells are able to grow, build, fall apart, and even reduce. Most of the time these reactions can occur because a chelating agent does or does not successfully bind to a cofactor in the enzyme. If a chelating agent successfully binds to its cofactor it keeps the cofactor from binding to the enzyme thus causing the enzyme to lose its shape and this will keep the reaction from occurring because the active site will not fit the substrate it needs. However, if the chelating agent does not bind to the cofactor this will allow the cofactor to then bind to the enzyme, this connection will then cause the enzyme to keep its respective shape. This will then allow the expected substrate to successfully bind to the enzyme's active site and the reaction will occur. In this experiment we will be testing how the combination of catechol and oxygen can cause the enzymatic reaction of catecholase which will result in benzoquinone and water, this is represented in the equation catechol + ½ O2 yields benzoquinone + H2O (Laboratory Exercises for Biology 101). This will be determined by observing how the binding of different chelating agents (EDTA, PTU, and citric acid) to certain cofactors (copper, calcium, or magnesium) in potato enzymes causes or keeps the enzymatic reaction catecholase from occurring. This will be measured by the amount of benzoquinone which is produced which can be proven by how much green light (540 nm) is reflected in the spectrometer, because catechol is clear which means it will not absorb and color and reflect no color (Laboratory Exercises for Biology 101). As benzoquinone is produced it turns the potato a orange color will then absorb green light which means that the more benzoquinone is produced the higher the reflected amount of green light will be(Laboratory Exercises for Biology 101). Our independent variables are the chelating agents because that is what can be changed, while the cofactors are the dependent variable since they cannot be changed. It is hypothesised that the chelating agents which bind to copper will yield the least amount of absorbance because copper is a cofactor naturally found in potatoes while calcium and magnesium are not.
Materials and Methods:
First, the catecholase enzyme solution was prepared with a chilled white potatoes, the potatoes were chilled in order to prevent the denaturing of the enzymes before the experiment began. Then, the chilled potato was peeled and half of the potato was chopped into pieces, the potato was peeled because its brown skin would begin to absorb green light prematurely which would mess with the experiments results. It was chopped so that blending would be smooth and easier and only half of it is used because if there is too much of the enzyme in the solution the reaction cannot be monitored correctly. After this the chopped potatoes were put into a chilled blender, the blender was cold so heat would be reduced and prevent the denaturing of the enzymes. 500mL of chilled distilled water into the blender as well, chilled distilled water is used to prevent the denaturing of the enzyme, and also because distilled water is hypotonic to enzymes thus giving it the ability to lyse the enzymes and ensure that there are no unwanted ions in the solution. Everything was blended together in three 10 second burst, to get the enzymes out of the starch which is inside the potato and to also prevent excessive heat which would denature the enzymes. After this 3 pieces of cheesecloth were stacked ontop of eachoter and the solution was poured through it and into a beaker, the cheesecloth is used to separate large pieces which can block the light in the spectrometer and mess up your readings. Finally, the solution was then poured from the beaker into a vial (filling it to the top), quickly sealed, and placed in ice, in order to prevent oxygen from entering the vial which would start the enzyme reaction before the experiment begins.
The control in this experiment was the vial which was given 2 mL of H2O instead of a cofactor, it acted as a standard absorbency value in the instance that the system works accordingly and was used as a comparative for the other absorbency values. First, turn on spectrometer 15 minutes before using it and set spectrometer to 540 nm, set the spectrometer early so that it will be ready when the first reading needs to be taken and set it to 540 nm because it corresponds to the color green. The spectrometer is set to a wavelength which corresponds to green light because the absorbance of green light is what will be measured during the experiment, as the experiment goes on catechol will be converted into benzoquinone which will turn the piece of potato orange thus making it absorb the green light. Then, 5 test tubes were obtained and each was labeled accordingly (test tube 1,2,3,4, and 5). Tube 1 got, 1 mL of enzyme solution, 2 mL of EDTA, and 2 mL of catechol were added after 10 minutes. Tube 2 got 1 mL put 1 mL of enzyme solution, 2 mL of PTU, and 2 mL of catechol were added after 10 minutes Tube 3 got 1 mL of enzyme solution, 2 mL of citric acid, and 2 mL of catechol were added after 10 minutes. Tube 4, the control, got 1 mL of enzyme solution, 2 mL of dH2O, and 2 mL of catechol were added after 10 minutes. Tube 5, the calibration tube, got 5 mL of dH2O. Each tube needed a total of 3mL of liquid not including the catechol solution, except the calibration tube (tube 5) because it will have 5 mL of dH2O, the catechol solution was added to tubes 1-4 10 minutes after its cofactor was put into the tube.
Prior to the beginning of readings each tube was shaken and gently flip every 2 minutes allowing the enzyme and chelating agent to mix and bind with their specific ions for 10 minutes. After these 10 minutes the catechol was added to tubes 1-4 and Parafilm was placed over the top of the test tubes . Tube 5 being the calibration tube (blank) was the first to be placed in the spectrometer to calibrate the machine before the other tubes were measured. Then the other tubes were inverted and placed into the spectrometer, their measurements were recorded. Tubes 1-4 were then placed into a water bath set to 25 degrees celsius to keep the enzymes temperature constant, and 10 minutes were allowed to pass before the next recording. After 10 minutes the tubes were taken out of the water bath, wiped with a Kimwipe, and inverted before recording the second reading. Once again the spectrometer was calibrated with Tube 5 before placing the other tubes in the spectrometer to be read and measurements recorded. Each tubes absorbable was recorded in the initial reading, and each tubes absorbance was again recorded after the 10 minutes of sitting in the 25 degrees Celsius, the color change observed was also recorded.
Results:
In consequence, the tube which had no chelating agent and contained dH2O instead (Tube 4), which was the control, had the most amount of absorbance with a value of 0.239 Absorbance (Table 2 & Figure 2). The tube which had the chelating agent EDTA (Tube 1), which binds to the cofactors calcium and magnesium, had the second highest amount of absorbance with a value of 0.071 A (Table 2 & Figure 2). The tube which had the chelating agent citric acid (Tube 3), which binds to the cofactor copper, had the second lowest amount of absorbance with a value of 0.057 A (Table 2 & Figure 2). The tube which had the chelating agent PTU (Tube 2), which binds to the cofactor copper, had the lowest amount of absorbance with a value of 0.035 A (Table 2 & Figure 2). Also, over time each tube's amount of absorbance went up. Tube 1 went from 0.263A to 0.334 which brought enough benzoquinone to change the color from clear to brown-orange, Tube 2 went from 0.124A to 0.159A which did not bring about enough benzoquinone to change the color so it stayed clear, Tube 3 went from 0.213A to 0.270A which did not bring about enough benzoquinone to change the color so it stayed clear, and Tube 4 went from 0.138A to 0.377A which brought enough benzoquinone to change the color from clear to a brownish (Table 1).
Discussion:
In the end, our hypothesis was supported because the chelating agents PTU and citric acid had the lowest amount of absorbance because it successfully bound to the cofactor copper. These results came about because the cheating agents bound to copper instead of catecholase which caused the enzyme to lose its shape which then kept the enzymatic reaction from occurring, which is why they had no color change (Figure 1). At the same time the enzymatic reaction which involves catecholase naturally has no chelating agents to prevent or slow down the reaction, this is why the tube with dH2O, our control, had the correct results because it had the highest amount of absorbency (Figure 1). It makes sense that the tube with EDTA as it chelating agent had the second highest amount of absorbency because calcium and magnesium are not cofactors which are naturally in potatoes enzymes, because of this EDTA had nothing to bind with thus causing the enzyme to keep its shape which allowed for the production of benzoquinone (Figure 1). The production of benzoquinone is the reason why the potato color changes and it will absorb more green light. The control legitimized the results of the experiment because they set the expected amount of absorbency for this enzymatic reaction by mimicking what could happen if this occurred naturally without any chelating agents, thus allowing us to accurately analyse whether or not the chelating agent bound to its respective cofactor or not.
One of our errors could have been the experiments set up, while or after pouring the enzyme solution into the vial it could have accidently exposing the vial of enzyme to oxygen prematurely which could have thrown off the readings a little bit. One last error could have been the temperature change of the tubes from when they were in the ice cooler and in the ice bath. In the cooler the ice was slowly melting which would increase the temperature of the solution when the first reading was taken. When the tubes were transferred to the ice bath which was a controlled machine where the temperature could neither increase nor decrease, then the second reading was taken. The enzymatic reaction could have sped up or slowed down because of the temperature difference thus giving us slightly inconsistent results. In the grand scheme of things, this experiment would really help potato farmers in the agriculture industry who sell their potatoes. Using preservatives which mainly consist of chelating agents like PTU or citric acid could increase their product's shelf life and fridge life. This would appeal to consumers because after a potato brown's most people will not want to buy or eat it, but if you cut a potato and it is almost still white the next day one will be more likely to use the rest and buy that kind of potato from that company again. After performing this experiment, I would like to know what other cofactors in potatoes could either help or hinder the activity of catecholase.