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We were able to measure enzymatic activity by the change in absorbency per second with a spectrophotometer. By testing different concentrations of peroxides and its reaction rate in seconds, we were able to see that as the amount of enzyme increased the catalytic reaction also increased. The optimal amount of peroxides concentration to be used in the subsequent experiments was determined to be 1.
0 ml. Any amount above this would have caused the rate of absorbency to be too fast, making it too difficult to get accurate readings.
Any amount below this would not have produced a reaction at an appreciable rate. ” (Dolphin, Fleck, Collect and Wastage, p. 76) In addition, our results show that a rise temperature and pH only increase the rate of reaction to a certain point before the reaction rate begins to decline dramatically. In the case of boiling of the enzyme there was no rate of reaction found whatsoever.
A similar result was found when hydroxylation was added to the peroxides and it caused an inhibition reaction.
Overall, the results show that the peroxides enzyme is sensitive with reference to the above factors in whether or not a reaction is catcalled. INTRODUCTION Enzymes are essential in the breakdown of certain materials or molecules that cannot be used by or are harmful to an organism as they are, into products that can be used or are not harmful for the organism.
They are proteins and their structure consists of amino acids with a specific shape. Enzymes have an area called an active site where substrates (only a particular molecule or material to be converted) bind.
When the substrate is bound to the active site on the whole entity becomes an enzyme-substrate complex. The substrate’s covalent bond is disrupted and this chemical change constructs a new product from the original abstract while leaving the enzyme unaffected. Once this new product is released, the enzyme can bind again with more of these molecules needing conversion. Sometimes the enzyme works with commences or cofactors such as vitamins or metallic ions to help the binding process.
In other cases competitive inhibitors are at work and prevent a substrate from being bound to the active site on the enzyme. The competitive inhibitor is similar enough to bind with the enzyme, but because it is not a perfect match, the enzyme then loses its ability to catalyst a reaction for that moment. In accordance with these properties, we ill see how certain factors affect the reaction rate of peroxides. For our purposes in this lab we used the enzyme peroxides extracted from a turnip.
Peroxides, along with the help of its iron ion cofactor, catalysts harmful hydrogen peroxide (H2O) into a harmless compound and water. In order to follow the rate of reaction for the breakdown of hydrogen peroxide, we used ecological, a colorless dye, which donates electrons and turns brown when it is oxidized. We used this dye so that we could measure the absorbency with the spectrophotometer as the hydrogen peroxide is being broken down and the lour change gets stronger over specific time intervals.
We developed several null hypotheses for these experiments: 1) The amount of enzyme added to the reaction will not affect the rate of reaction. 2) Temperature will not affect the enzymatic activity. 3) pH will not affect enzymatic activity. 4) Similar molecule to substrate will not affect enzymatic activity. MATERIALS AND METHODS Materials and methods are taken from Lab Topic 7 in the Biological Investigations, 9th Edition. RESULTS Graph 1- Effects of Peroxides Amounts Graph 2 – Temperature Effects on Peroxides Activity
Graph 3 – pH Effects on Peroxides Activity Graph 4 – Boiled Peroxides Results Graph 5 – Hydroxylation Results Graph 6 – Optimum Temperature for Reaction Rate of Peroxides Graph 7 Optimum pH for Reaction Rate of Peroxides In Graph 1, Effects of Peroxides Amounts, it shows the difference in rates of reactions with different concentrations of peroxides in the solution Tubes 2 & 3 at 0. 5 ml, Tubes at 1. 0 ml and Tubes 6 & 7 at 2. 0 ml, along with corresponding line slopes. Graph 2, Temperature Effects on Peroxides Activity, shows the difference in rates of reaction for 1. Ml peroxides at ICC, Room Temperature ICC, ICC and ICC along with their corresponding line slopes. For Graph 3, pH Effects on Peroxides Activity, it shows the rates of reaction at pH 3, pH 5, pH 7 and pH 9, along with its corresponding line slopes at 1. 0 ml peroxides. Graph 4, Boiled Peroxides Results, shows the rates of reaction for 1. 0 ml between a Normal Extract of peroxides and a Boiled Extract of peroxides, both with their corresponding line slopes. In Graph 5, Hydroxylation Results, it shows rates of reaction for 1. Ml between a Normal Extract of peroxides without hydroxylation and a Hydroxylation-treated Extract of peroxides. Graph 6, Optimum Temperature for Reaction Rate of Peroxides, shows the slope of the line at each temperature. Lastly, Graph 7, Optimum pH for Reaction Rate of Peroxides, shows the slope of the line at each level of PH. DISCUSSION The significance of the observations in testing the enzyme peroxides and its rate of breaking down hydrogen peroxide helped us to make a decision as to whether to accept or reject our hypotheses in the experiment.
The amount of peroxides concentration had a direct relationship to how slowly or how quickly a reaction took place. This result allowed us to reject our hypothesis that the mount of enzyme added to the reaction will not affect the rate of reaction. This test was important so that we could ascertain the best amount of concentration to use in the subsequent experiments with the spectrophotometer set at absorbency 470 NM and timed recordings at 20-second intervals for a total of 2 minutes. At 0. 5 ml of peroxides the reaction time was too slow thus no appreciable line or slope was rendered to measure the reaction with any accuracy.
Conversely, it was a challenge to get accurate absorbency readings at 2. 0 ml of peroxides because the pace of the reaction appreciated so quickly and hen met equilibrium. At 1. 0 ml of peroxides the reaction time rendered an appreciable line and slope making it easier to record the absorbency every 20 seconds for 2 minutes and ultimately, the best concentration for use in the next experiments. It is known that when heat is applied to molecules, they move faster and collide more as the temperature rises.
This is also true for the enzyme peroxides and its substrate until the temperature reaches ICC and then the reaction begins to taper off and it dives down drastically at ICC when the hydrogen bonds holding peroxides structure together begin to break. The results of this test confirm the same by the slope of each line and thereby we are able to reject our hypothesis that temperature has no effect on peroxides rate of reaction. At ICC the slope of the line is 0. 0071, at Room Temperature ? ICC the slope is 0. 0094, at ICC the slope is 0. 091 and at ICC it is 0. 0052. This is easily seen on the derivative Graph 6: Optimum Temperature for Reaction Rate of Peroxides attached. It should be mentioned here that in class it was decided from the table of results on the board for this experiment that ICC was the optimal temperature. In addition, it is difficult to fully see the results from the line graph number 2. As we can see here from the derivative graph, that just numbers without the proper graphs can be misleading and it is necessary to take further steps in clarifying the observations and results.
As for pH effects on peroxides activity, Graph 3, indicates that the amount of acidity or bassinets to a solution changes the three-dimensional structure of the enzyme and thereby changes the ability to bind with the substrate in an effective manner. Here we tested the null hypothesis: pH will not affect enzymatic activity. The results from Graph 3: pH Effects on Peroxides Activity indicate that the more acidic pH 3 level disrupted the enzyme’s ability to bind with its substrate and its reaction rate did not appreciate noticeably.
As the solution became less acid at pH 5, the greatest reaction efficiency resulted. Once the pH was at 7 and beyond the reaction rate for peroxides and its binding ability became poor and the reaction rate declined. Again, it was necessary to do a derivative graph to see the slope results clearly. In Graph 7: Optimum pH for Reaction Rate of Peroxides, the rate of reaction increased drastically from pH 3 with a slope f 0. 00007 to pH 5 with a slope of 0. 0055 and then trails off as the bassinets increases at pH 7 with a slope of 0. 047 and plummets as it reaches pH 9 with a slope of 0. 0022. We can see the optimal pH is 5 in this experiment and we were able to refute our null hypothesis because it was shown that pH does affect peroxides reaction rates. In boiling the peroxides extract, the result was that no reaction activity was found. As mentioned in our book by Dolphin, Fleck, Collect and Wastage, the enzyme is denatured because the high temperature broke a large number of hydrogen bonds, which dramatically changed the structure of the enzyme permanently (82).
A denatured enzyme cannot catalyst a reaction and this result appears evident on Graph 4: Boiled Peroxides Results. The comparison between a normal extract of peroxides and a boiled extract of peroxides shows that zero absorbency with no appreciation was yielded over the two minutes for the boiled extract as opposed to the normal extract that did yield an appreciable line with a slope of 0. 0099. Again, we can refute our null hypothesis of temperature will not affect the enzymatic activity. Finally, a null hypothesis that a similar molecule to substrate will not affect enzymatic activity was tested.