Catalase Titration Lab

Catalase Titration Lab

In this lab, we were using enzymes (yeast) to break down hydrogen peroxide. But after a set time (10 seconds, 30, 60, 180, 360), we injected sulfuric acid to stop the catabolic process. We then titrated a 5 ml sample of the solution with potassium permanganate to see how much hydrogen peroxide was left to consume the potassium permanganate in the 5 ml solution. This experiment consisted of three different parts. 

Part B: In this section of the experiment, we determined the amount of hydrogen peroxide (H2O2) initially present in a 1.5% solution without adding a catalase, or enzyme, to the reaction mixture. The amount we find will be our base line, or constant, for the upcoming parts of the experiment. 

                 We started this experiment by measuring out 10 ml of the 1.5% hydrogen peroxide into a plastic cup. (Insert photo) Then, we added a mL of water. For this section, we added water instead of an enzyme solution in order to find constant data that we could compare to the data of the solutions that contained enzymes. After this we added 10 mL of sulfuric acid and mixed the solution. This stopped the reaction by denaturing the enzymes. Next we removed a small sample of the final solution and placed it into the titration cup. As might have guessed, we then titrated the solution. This means that we added small amounts of potassium permanganate until the solution permanently changed to a light pink or brown color. We compared the initial amount of potassium permanganate in the burette to the final amount in order to find out how much potassium permanganate was needed to discover how much hydrogen peroxide was used up. 

Part C: In this portion of the lab, we wanted to calculate the rate at which H2O2 turns into H2O and O2 (or decomposes) in an uncatalyzed reaction. An uncatalyzed reaction simply means that an enzyme was not present. Additionally, this results in the activation energy of the reaction to be higher. After the solution was left to sit uncovered for twenty-four hours, it was then tested to gather this information.

      Natalie made a great video for the paragraph below

To prep for the experiment, we first placed about 15mL of H2O2 in a beaker and let it sit over night. After, we added 1mL of H2O (also known as, water) because we were calculating data for an uncatalyzed reaction. Next, we added 10 mL of H2SO4 (also known as, Sulfuric Acid) to the solution. If the solution we were testing had an enzyme, the acid would denature the enzyme since it is a protein. The enzyme would denature because the acid would lower the pH of the solution and enzymes have a set pH level in which they can function normally. Then, we mixed the contents of the beaker and drew a 5 mL sample of the solution. We took this 5mL sample and added KMnO4 (also known as, the titrate in this experiment) drop by drop, to measure the amount of  H2O2 still remaining in the solution. During this step, we also swirled our sample to insure that the titrate distributed evenly. It only took one drop of KMnO4 to give us the data that we needed for this experiment. Below is the data collected from this portion of the experiment.

                

Part D: In this part of the lab, we wanted to figure out how much H2O2 was eaten up depending on how long we left the H202 to react with a catalase. In this case we used yeast! We noticed a lot from the results when looking at the different amount of times we left the H2O2 with the yeast.       

When we prepared this we had five different beakers that each had a time label. "10 seconds, 30, 90, 180, 360". Each time label represented how long we would let the H2O2 react with the yeast before we applied 10 ml of sulfuric acid in order to stop the process. We added 10 ml of H2O2 and then 1 ml of yeast but the second that we added the yeast, we immediately started timing accord to the label on the beaker. After waiting, we add the acid and then extracted a 5 ml sample from the beaker. Proceeded to place it under the burette and calculated how much potassium permanganate it took to turn the sample a slight pink showing that there wasn't anymore H2O2 to react and "erase potassium permanganate.

 We noticed from our results (after graphing the data) that the longer we left H2O2 to sit with the yeast, the less amount of potassium permanganate was needed to turn the sample a slight pink. Showing that the catalase (yeast) was reacting with the hydrogen peroxide and reacting to make H2O and oxygen.

OUR DISCUSSION

-

So in order to determine the course of an enzymatic reaction, we had to measure out how much substrate was disappearing over certain time intervals. So we recorded our data for the amount of hydrogen peroxide used in every test, and made a graph to better depict this information. As you can see below, (insert graph here) the rate of the amount of H2O2 in a certain amount of time is the highest between 180 and 360 seconds. This means that the difference of the amount of hydrogen peroxide used up is greatest between 180 and 360 seconds, most likely because that is the biggest time interval. On the other hand, the smallest rate is between 0 and 10 seconds. This is because it is the shortest gap of time, so hydrogen peroxide does not have nearly the same amount of time to become used up as it does between the 180 and 360 second gap.

CONCLUSION

-

Overall, our group experienced a hands-on lab that taught us more about enzyme activity. The rate of the reaction had to do with the substrate concentration. As the concentration went higher, the reaction rate increased as well. At one point, the rate stays the same since the enzymes are always loaded with what they are catalyzing. In order to collect the right data on the reaction rate, we used an acid to denature the enzyme, and then titrate to see how much substrate was left over. In order to better this part, we could have been more precise in our titration. Other than that very tiny error, this lab went very smoothly!

This was our enzyme catalysis lab, hope you enjoyed reading!