Title: Enzyme Catalysis Lab

Introduction: Enzymes are proteins produced by living cells and act as catalysts in biochemical reactions. A catalyst affects the rate of a chemical reaction. The substrate, the substance to be acted upon, binds reversibly to the active site of the enzyme. Any substance that blocks or changes the shape of the active site affects the activity of the enzyme. Salt concentration is one way out of the following that enzyme action may be affected. If the salt concentration is close to zero or is very high, the enzyme will precipitate. Many enzymes perform best in the neutral pH range and are denatured at a higher or lower pH. Chemical reactions generally speed up as the temperature is raised. However, there is a temperature optimum of enzyme-catalyzed reactions. Furthermore, activators increases the rate of a reaction and an inhibitor decreases the reaction rate. The purpose of this experiment is to calculate the rate of reaction and how much substrate disappears over time in an enzymatic reaction.

Hypothesis: If the conversion of hydrogen peroxide to water and oxygen by the enzyme catalase is observed, then the amount of oxygen generated can be measured and the rate of the enzyme-catalyzed reaction can be calculated.

Materials: The following chemicals are needed for this experiment: 1.5% H2O2 (hydrogen peroxide), H2SO4 (sulfuric acid), at least 400 ml of catalase, and 2% KMnO4 (potassium permanganate). There should also be distilled water. A pipette should be used to add water and at least 3 separate syringes should be used to avoid contamination. There should be at least eight beakers or cups to perform the titrations, for the reaction mixture, the chemicals, and for distilled water.

Procedure: First, the baseline had to be established, which tells how much H2O2 was in the initial 5 ml sample. To establish a base line, 10 ml of 1.5% H2O2 was added into a glass beaker. Instead of enzyme solution, 1 ml of water was added. Then, 10 ml of H2SO4 was added and the solution was mixed well. A 5 ml sample of this solution was transferred into another beaker and assayed for the amounted of H2O2. A syringe was used gather 6 ml (the initial reading) of KMnO4 , which was then added one drop at a time to the solution until it turned to a persistent pink or purple color. The solution was gently swirled after each drop. After the solution turned to a persistent pink or purple, the final reading of the syringe was noted and the amount of KMnO4

used could be calculated. The amount of H2O2 that was in the baseline can also be calculated.

To measure the enzyme-catalyzed rate of H2O2 decomposition, it should be determined how much H2O2 had been consumed after 10, 30, 60, 90, 120, 180, and 360 seconds. Starting with the amount of substrate decomposed after 10 seconds, 10 ml of H2O2 was put into a beaker. 1 ml of catalase was added and the solution was swirled gently for 10 seconds. As soon as the catalase was added, the timer was started and as soon as the timer hits 10 seconds, 10 ml of H2SO4 was added to stop the reaction. Then a 5 ml sample of the solution was removed and KMnO4 was added a drop at a time until a persistent pink or purple color was obtained. The final and initial

reading of KMnO4 was recorded for calculation purposes. This procedure was repeated for 30, 60, 90, 120, 180, and 360. This lab group (results below) was responsible for the rate of reaction at 60 and 90 seconds.

Results:

Baseline calculation: (Final reading of syringe: 0 ml) – (Initial reading of burette: 6 ml) = 6 ml KMnO4 added to baseline. The amount of KMnO4 used is proportional to the amount of H2O2

present in the baseline. The class average for base line is 5.4375.

Lab Group Data

KMnO4 (ml) 60 Seconds 90 Secondsa) Base line 6 ml 6 mlb) Final reading 1.2 ml 1.1 mlc) Initial reading 5.8 ml 5.8 mld) Amt of KMnO4

consumed (B minus C)4.6 ml 4.7 ml

e) Amt of H2O2 used (A minus D)

1.4 ml 1.3 ml

Class Data

Time (sec) H2O2 Used (ml)10 1.430 0.460 0.590 0.5120 2.5180 -0.1360 2.3

1. Determine the rate of reactions between each of the time points. (Any 2 points of the curve: divide the difference in the amount of product formed between these two points by the difference in time between them).

Time Intervals:

Initial 1-10

10-30 30-60 60-90 90-120 120-180 180-360

Rate (µmoles/sec)

0.14 -0.05 0.003 0 0.067 -0.043 0.013

2. When is the rate the highest? The rate was the highest in the initial interval (1 second to 10 seconds). It was predicted to have a higher rate near the 10-30 or 30-60 range or near the temperature optimum.

3. When is the rate the lowest? It had a negative rate between the 10-30 range. This data doesn’t correlate with what was expected of the 10-30 range. The rate of reaction should have at least been a positive rate.

4. Explain the inhibiting effect of sulfuric acid on the function of catalase. The sulfuric acid lowers the pH and denatures the enzyme to stop the enzyme’s catalytic activity. The enzyme no longer conforms to the substrates.

5. Predict the effect that lowering the temperature would have on the rate of enzyme activity. The rate of reaction will decrease. Lowering the temperature usually slows down the rate of activity. Raising the temperature will disrupt their conformations.

6. Design a controlled experiment to test the effect of varying pH, temperature, or enzyme concentration. There should be a separate experiment for a neutral or optimal pH level, optimal temperature, and different amounts of enzyme added.

0 50 100 150 200 250 300 350 400-0.5

0

0.5

1

1.5

2

2.5

3

Enzyme-Catalyzed H2O2 Decomposition: Class Data

Time (Seconds)

H2O

2 De

com

positi

on (m

l)

Sources of Error: The class data has almost no correlation. There were many errors in the lab, probably due to misunderstanding of instructions and miscalculations. This data is not reliable due to the errors.

Discussion and Conclusion: The hypothesis was supported in this experiment. The overall class data isn’t considered reliable or very logical. However, the purpose was fulfilled. The baseline tells how much H2O2 was in the initial 5 ml sample. The difference between the initial and final readings of KMnO4 tells how much H2O2 is left after the enzyme-catalyzed reaction. More H2O2

should remain if the reaction time is shorter. After doing the timed experiments to measure the amount of substrate decomposed, the class results were unexpected. A more correlated curve was expected, but it turned out to be uncorrelated and included a negative reaction rate. The enzyme should have had the highest reaction rate at the temperature optimum. The temperature was room temperature in this experiment. If that statement is correct, then the data gathered at 120 seconds should be the temperature optimum and the fastest rate of decomposition. The rate should have gradually increased from the initial value to the temperature optimum, but the data gathered showed that it didn’t. The pH wasn’t calculated, so that may have been a factor in the overall rate of reaction. Salinity was also not measured. This experiment may need to be redone or a controlled experiment needs to be done to double check if the uncorrelated rates of reaction were a result of human error, or if this was what was supposed to happen.