ObjectiveObjectives of this lab include to study: the effect of Catalase on the substrate hydrogen peroxide, the effect of pH on enzyme activity, the titration of hydrogen peroxide in order to deduce the amount of it in an designated amount of solution, the rate of reaction of the enzyme Catalase, and how time effects the total consumption of hydrogen peroxide.

IntroductionEnzymes are special proteins that are produced by all living cells. Enzymes act as catalysts in biochemical reactions by changing the rate of the reaction. Catalysts work by binding to substrates to perform certain functions: joining monomers, breaking polymers into monomers, moving functional groups and atoms, or transferring electrons A common catalyst found in the body is Catalase, and its purpose is to maintain a comfortable level of hydrogen peroxide for its respective host. The general reaction that Catalase works with is 2 H2O2 -----> 2 H2O + O2. Many factors can inhibit Catalase from working. Inhibitors such as salt concentration, pH, and temperature can denature an enzyme. Such is the case with Sulfuric Acid and Catalase where the acid increases the pH enough to make the enzyme denature. Potassium permanganate is a powerful oxidizing agent that can decompose certain compounds. KMnO4 decomposes hydrogen peroxide through the following reaction 5H2O2 + 2KMnO4 + 3H2SO4 ----> K2SO4 + 2MnSO4 + 8H2O + 5O2. Many things are ascertained through measuring kinetics of reactions including the amount of product formed, or the amount of substrate used. A graph can be

constructed by analyzing the how much substrate is converted into product. It is possible to determine the amount of substrate converted by recording the amount of KMnO4 that is used up when titrating as long as the KMnO4 is 2.5 times more concentrated than the hydrogen peroxide. Once the data is recorded, it can be made into a graph. When looking at the graph the rate of reaction can be deduced by determining the slope of the line between each point. The rate of reaction can tell us how fast the reaction works and many other useful things.

ProcedureDay One: All materials needed for the lab were collected. These materials were: Hydrogen peroxide, 1M sulfuric acid, potassium permanganate, water, two glass beakers, a graduated cylinder, a pipette, a ring stand, a syringe, and a burette. 1. 2. I clamped the burette onto the ring stand, and filled the burette one-quarter full with potassium permanganate. I measured 10 mL hydrogen peroxide in the graduated cylinder and emptied it into one of the glass beakers.

3. I collected 10 mL sulfuric acid with the syringe and emptied it into the same beaker as the hydrogen peroxide. 4. I collected 1 mL water with the pipette and emptied it into the same beaker. 5. Using the syringe, I transferred 5 mL of the total solution to the second glass beaker. I placed the beaker under the burette. 6. I recorded the initial amount of permanganate in the Baseline Calculation table. I used the permanganate in the burette to titrate the hydrogen peroxide in the second glass beaker. 7. Once the contents of the second glass beaker changed color to a light pink, I stopped the titration. 8. I recorded the final value for permanganate. I subtracted the initial value from the final value and obtain the amount of hydrogen peroxide that was titrated. I recorded both values in the Baseline Calculation table(table 1). 9. I dumped the contents of both beakers into waste beakers. I returned excess permanganate to its vessel. I washed all the materials used and put them back in their places.

Day Two: The same materials from day one were collected; water was replaced with chilled catalase, an enzyme that catalyzes the decomposition of hydrogen peroxide. Some sort of timekeeping device was also needed. 10.I clamped the burette onto the ring stand and filled the burette with potassium permanganate. 11.I measured 10 mL hydrogen peroxide with the graduated cylinder, and then emptied it into one glass beaker. 12.I obtained 10 mL sulfuric acid, using the syringe. 13.I obtained 1 mL of the catalase using the pipette, and emptied it into the same container as the hydrogen peroxide. 14.I used the timer to count out 10 seconds, then emptied the sulfuric acid into the contained containing the catalase. 15.I used the graduated cylinder to measure 5 mL of the entire solution. I emptied the 5 mL into the second glass beaker. 16.I recorded the initial value for permanganate in table 2. I titrated the contents of the second glass beaker with the permanganate, and recorded the final value on the burette. 17.I subtracted the initial reading from the final reading to determine how much hydrogen peroxide was titrated by the permanganate. I subtracted this amount from the initial amount of hydrogen peroxide to determine how much hydrogen peroxide was catalyzed by the catalase. I recorded both values in table 2. 18.I emptied all excess solution into the waste beakers, and clean the tools I had used. 19.I repeated steps 11 to 18, but varied the length of time that the peroxide reacted with the catalase (step 14). I repeated these steps for lengths of 30, 60, 120, 180, and 360 seconds. I recorded all data I obtained in table 2. 20.I cleaned and stored all materials that I used for the procedure. 21.My instructor gave me the ideal data to compare my results with. 22.I graphed the data that I had gathered and the ideal results data. 23.I found the slopes between each point of the Ideal Graph and the Actual Results graph. I then recorded those slopes in tables 4 and 6.

Analysis (Data)

Table 1 Baseline Calculation Final Reading of Burette Initial Reading of Burette Baseline (Final Initial)

42.5 mL 49.9 mL 3.4 mL KMnO4

Table 2Time(seconds)

KMnO4 (mL) A. Baseline B. Final Reading C. Initial Reading D. Amount of KMnO4 consumed ( B minus C) E. Amount of H2O2 used (A minus D)

10 3.4 32.5 31 1.5 1.9

30 3.4 36.0 32.6 3.4 0

60 3.4 42.8 39.4 3.4 0

120 3.4 43.2 43 0.2 3.2

180 3.4 43.3 43.2 0.1 3.3

360 3.4 43.5 43.4 0.1 3.3

Data Table for Actual Results 3

Time(se c) Amount of H202 consumed(mL) 0 0 10 1.9 30 0 60 120 180 360 0 3.2 3.3 3.3

Actual Rate's Data Table 4 Time(sec) Rates(ml/sec) Initial 0 to 10 0.19 10 to 30 - 0.095 30 to 60 0 60 to 120 0.053 120 to 180 0.0016 180 to 360 0

Ideal Results Data Table 5

Time(sec) Amount of H202 consumed(mL) 0 0 10 1.9 30 2.4 60 2.6 120 2.9 180 2.9 360 2.9

Ideal Result's Rate Data Table 6Time(sec) Rates (mL/sec) Initial 0 to 10 0.19 10 to 30 0.025 30 to 60 0.0066 60 to 120 0.005 120 to 180 0 180 to 360 0

Conclusion

When we titrated our baseline solution of KMnO4 we found that the maximum amount of hydrogen peroxide in a five milliliter solution is 3.4 milliliters ( refer to table 1). We used 3.4 as the point of reference for maximum enzyme activity. We started by assuming that when Catalase had not had any time to work on the reactants and that no reactants were converted into product. For our first test result, we let the Catalase function on the H2O2 for 10 seconds. When we titrated, the sample had an initial buret reading of 32.5mL and a final reading of 31.0mL. The color of the solution was a slight pink. We found that the Catalase converted 1.9mL of hydrogen peroxide into product by subtracting the initial reading of the buret from the final reading to get 1.5mL. We took the maximum amount of hydrogen in a sample minus the 1.5mL to get the 1.9mL of product produced. When we allowed the Catalase to function on the hydrogen peroxide for about 30 seconds and we titrated the sample, we found that there was still 3.4 milliliters of hydrogen present. To ascertain that there was still 3.4mL in the sample we took the initial subtracted from the final amount. We took that amount and subtracted it from 3.4mL and the result was 0mL. When we allowed the Catalase to work on the hydrogen peroxide for 60 seconds and titrated the sample until a slight pink, we found that the Catalase again had not consumed any substrate. We pondered these two results and realized we had made a huge error. While washing the lab tools between each titration we did not wash a graduated cylinder. The graduated must have retained some of the sulfuric acid thus denaturing the Catalase. After those two data points, our group began to clean out everything in between experiments. The Catalase was then allowed to react with the hydrogen peroxide for 120 seconds. After 120 seconds, our group denatured the enzyme with a solution of sulfuric acid. We took an initial buret reading of 43mL and titrated until the solution was a slight pink. We

recorded the final reading to be 43.2mL. We subtracted the 43mL from 43.2mL and found that only .2mL of hydrogen peroxide was left. When compared to the total amount of hydrogen peroxide in the 5mL solution we ascertained that the enzyme converted 3.2mL of the total 3.4mL into products. The Catalase was then allowed to react for 180 seconds with the hydrogen peroxide. Our initial buret reading was 43.2mL and we titrated. With only a couple drops of KMnO4, our solution turned pink and we took the final buret reading of 43.3mL. We subtracted the 43.2mL with the 43.3mL and found that .1mL of hydrogen peroxide was left. We then subtracted . 1mL from the maximum amount of hydrogen peroxide in a sample and found that the Catalase converted 3.3mL of hydrogen peroxide into product. For our last experiment, the Catalase was allowed to react with the substrate for 360 seconds. After the 360 seconds, 10mL of sulfuric acid was added to denature the Catalase; thus, the reaction stopped. The initial reading of the buret was 43.4mL and the solution was titrated until a slight pink. The final reading was 43.5mL. We took the 43.5 minus the 43.5mL and found that only .1mL of hydrogen peroxide remained in the solution. We took the maximum amount of hydrogen in a sample, 3.4mL, and subtracted the .1mL. We got 3.3mL and deduced that the Catalase converted 3.3mL of hydrogen peroxide into product. After finding and recording our results we graphed them (refer to the Actual Results: Amount of H202 consumed by Catalase for Different Time Intervals of Catalase Activity). We took the amount of substrate consumed by the Catalase for each point in time ( refer to data table 3). When looking at the graph in context one deduces that the longer Catalase was allowed to react with the substrate there was more substrate converted into products. We took each point of Graph Actual Results: Amount of H202 consumed by Catalase for Different Time Intervals of Catalase Activity and found the slopes between them,

refer to data table 4 for the results. This slope graph tells us that as the reaction went on less and less hydrogen peroxide was consumed. This is explained by simple reasoning, if there was only 3.4mL of hydrogen peroxide in the solution as the Catalase reacted there was consistently less and less substrate to react with. Therefore, as there is less substrate to react with, the rate at which substrate is converted into product will decrease. When we compared our results to the ideal results, we determined that most of our results were accurate. This does not include the results that our group did not clean the graduated cylinder. Our results and the ideal results only differ by a few points for their respective graphs. When we take both graphs into context, we see that the longer the Catalase reacted, the more products produced. Furthermore, when we juxtapose both rate tables, 4 and 6, we see that in general as the reactions with the Catalase were given more time to react the rate at which the Catalase reacted with substrate decreased.

Carlton Christie Mrs. Mountz 10/13/10

Enzyme Catalysis Lab