Cool Enzyme Lab

Part 2B:

In this part of the experiment we determined the baseline amount of H2O2 present in a 1.5% solution. We didn't add any enzymes to this reaction because this initial value that we will get is essentially the control group and so we need to know how much H2O2 was initially present in the solution.

First, we took about 10ml of 1.5% H2O2 and poured it into an empty cup. Then we added 1ml of H2O as a replacement for the enzymes. We need the solution to remain uncatalzyed so that our control value can be as accurate as possible, and so that is why we did not add the enzyme into the solution. After that, we took about 10ml of H2SO4 and poured it into the solution and mixed it all together.

In the picture above we are using a pipette to put 10ml of H2SO4 into the solution.

After we mixed the solution, we took a 5ml sample of it and we slowly added KMnO4, with a burette, to the solution until a pink color was obtained and stayed pink. The amount of KMnO4 used is directly proportional to the amount of H2O2 that was in the solution. When the solution turns pink that means the amount KMnO4 is exactly the amount of H2O2 in the solution because there is enough of each to react with each other.

In the photo above we are using the burette to slowly add drops of KMnO4 to the solution until it turns pink. Before we start to add the KMnO4 we take the initial reading of the burette and after the solution turns pink we take a final reading. If we subtract final-initial we get the total amount of KMnO4 used, thus getting the amount of H2O2 in the solution.

The figure above shows our final and initial readings of the burette. After our calculations we concluded that there was 3.5ml of KMnO4 present and therefore found the amount of H2O2 that was in the solution.

In conclusion, we found that 3.5 ml is our baseline value and that is the value that the rest of our trials will be compared to.

Part 2C:

In this procedure, we are determining the rate of spontaneous conversions of H2O2 to H2O and O2 in an uncatalyzed reaction. To find the data needed, we first needed to take 15mL of H2O2 and let it sit uncovered for 24 hours. Then we repeat the steps seems in 2B. We took 10mL of the H2O2 into a new beaker and added 1 mL of H2O (instead of the enzyme solution) and 10 mL of H2SO4. We mixed it well and then added the KMnO4 titration. Surprisingly enough, it only took less than five drops of the KMnO4 to make the solution pink. 

The image above shows the data collected. It only took .1 mL of KMnO4 to make the solution pink and 3.4 mL of the H2O2 was spontaneously decomposed; 97.14% was decomposed in 24 hours. What does this mean? By letting the solution settle overnight for 24 hours, the majority was decomposed naturally and only leaving a small amount left. This is why it took only a few drops of KMnO4. The less amount of H2O2 you have, the less amount of KMnO4 you need since both need to have equal quantities to keep the solution pink. 

Part 2D:

For this section, our goal was to observe the effect of time on the enzyme-catalyzed reaction that converts hydrogen peroxide into water and oxygen. We used the base line assay from Part 2B as a sort of control group. This showed us the original concentration of hydrogen peroxide in the solutions if the reaction were not permitted to occur. For the rest of the reactions we used the following method. We measured out 7 quantities of 10 mL of 1.5% hydrogen peroxide (the substrate for our enzyme), added 1 mL of catalase (an enzyme in yeast) to each quantity of hydrogen peroxide, and allowed the catalyzed reactions to persist for varying amounts of time (10, 30, 60, 90, 120, 180 and 360 seconds), after which we would stop the reactions by adding 10 mL of sulfuric acid. The acid reduced the pH of the solution, which denatured the enzymes by changing their shapes and behavior, which meant that they were no longer capable of catalyzing the reaction. For each solution, we performed a titration with potassium permanganate, wherein we slowly added the titrate to the solution until it remained pink or brown, at which point the volume of titrate equalled the volume of hydrogen peroxide present in the solution. Our results are in the table below...

The result, as we expected, was that the longer we permitted the reactions to be catalyzed, the less hydrogen peroxide would be left, as more of it would have been converted to water and oxygen. Here's the same information again in a graph of amount of hydrogen peroxide (substrate) used up in the reaction versus time. Note that it is not the amount of hydrogen peroxide left over but the amount that was used up...

In this graph, the data looks a little rough, but it gets the point across: the longer a catalyzed reaction is allowed to persist, the less substrate is left.

All in all, this lab gave us the chance to observe enzymes in action and understand their behavior in reactions. Understanding them in a lab environment like this gave us a better idea of how they function in the context of our bodies to regulate the rate of the reactions that sustain us.