Problem: Before the lab, one should understand: •The general functions and activities of enzymes; •The relationship between the structure and function of enzymes •The concept of initial reaction rates of enzymes; •How the concept of free energy relates to enzyme activity; •That change in temperature, pH, enzyme concentration, and substrate concentration can affect the initial reaction rates of enzyme-catalyzed reactions; and •Catalyst, catalysis, and catalase After the lab, one should be able to:
•Measure the effects of changes in temperature, pH, enzyme concentration, and substrate concentration on reaction rates of an enzyme-catalyzed reaction in a controlled experiment; and •Explain how environmental factors affect the rate of enzyme-catalyzed reactions. Hypothesis: When one mixes catalase and hydrogen peroxide with sulfuric acid for a short period of time, there is a high rate of increase for the decomposition of hydrogen peroxide to hydrogen and water.
However, after one mixes these substances together for a longer period of time than necessary, substrate becomes overly-saturated in the solution which causes the enzyme not to work properly. Materials: •Goggles •Reaction vessels (cups) •Gloves •Syringes •Plastic Wrap •Rubber Bands •Potassium Permanganate •Sulfuric Acid •Catalase Extract •Hydrogen Peroxide Procedure: First, we made the base line assay to determine the fluctuation from the normal reaction mixture without catalase. This was the index of the initial concentration of H2O2 in solution. To establish a baseline, start by putting 10 mL of 1.
5% H2O2 into a clean glass beaker. Add 1 mL of H2O (instead of enzyme solution). Add 10 mL of H2 SO4 (1. 0 M). Mix well. Remove a 5-mL sample. Place this 5-mL sample into another beaker and assay for the amount of H2O2 as follows. Use a syringe to add KMnO4 , a drop at a time, to the solution until a persistent pink or brown color is obtained. Remember to gently swirl in the solution after adding each drop. Check to be sure that you understand the calibrations on the syringe. Record your reading. Proceed now to Exercise 2D: An Enzyme-Catalyzed Rate of H2O2 Decomposition.
In this experiment, we determined the rate at which a 1. 5% H2O2 solution decomposes when catalyzed by the purified catalase extract. To do this, we determined how much H2O2 had been consumed after 10, 30, 60, 90, 120, and 180 seconds. For the 10 second reaction first put 10 mL of 1. 5% H2O2 in a clean 50-mL glass beaker. Add 1 mL of catalase extract. Swirl gently for 10 seconds. At 10 seconds, add 10 mL of H2SO4 . For the remaining times, repeat these steps, with the reactions proceeding in the appropriate times. Each time, remove a 5-mL sample and assay for the amount of H2O2 in the sample.
Use a syringe to add KMnO4, a drop at a time, to the solution until a persistent pink or brown color is obtained. Record data. Results (Data): See Packet. We observed that the rate was at an increasing slope in the beginning, but it eventually leveled off due to the excess substrate molecules. Conclusions: To determine the rates of the reactions, we divided the difference in the amount of product formed between in the different time intervals between the time intervals. The first couple of intervals definitely had the highest rates in the experiment, 0. 01 and 0. 095, respectively.
This was because there were larger amounts of substrate molecules than enzyme molecules in the smaller time limits. We found that the longer the catalase reacted, the lower the reaction rate was because there were more product molecules as result of the chemical reactions between the enzyme and the substrate. From the interval 120 to 180 seconds, the rate was 0. The excess peroxide solution was what caused the purple color to surface in the solution. This information led us to accept the hypothesis that once there is excess substrate and not enough enzymes, the substrate molecules just stay in their initial state.
We learned that this situation comes to us in real life with intoxication. When someone drinks a certain amount of alcohol, it is reduced properly with catalase in the cells. However, when someone drinks too much, too many substrate molecules surface, and the catalase is not enough to break down all the alcohol. The possible error that might have occurred in our results was the effectiveness of the catalase. It may have not worked properly in our experiment, which is why we got a flat amount of permanganate used.