How To Calculate Enzyme Activity

Transcription

1 Exercise 6 - Enzyme Activity Introduction Enzymes are biological catalysts that regulate the rate of chemical reactions. Their 3-dimensional conformation and therefore their function can be affected by several variables. In this laboratory exercise, you will manipulate various factors that affect an enzyme s activity. The enzyme is catalase, which is found in most all living organisms. Catalase decomposes hydrogen peroxide (H 2 O 2 ), a toxic compound, into water and oxygen: 2H 2 O 2 + catalase 2H 2 O + O 2 The amount of oxygen created is directly proportional to the rate of the enzymatic reaction. Therefore, measuring the amount of oxygen produced provides a measure of the speed at which the reaction is proceeding. The effect of external factors such as substrate concentration, temperature, and ph will be examined. Materials Equipment test tubes and racks metric rulers graduated cylinders droppers marking pens (Sharpies) thermometers ice water bath warm (40 C) water bath hot (95 C) water bath Reagents and Solutions catalase (sheep s blood) 3% hydrogen peroxide (H 2 O 2 ) ph 3 buffered H 2 O 2 solution ph 5 buffered H 2 O 2 solution ph 7 buffered H 2 O 2 solution ph 9 buffered H 2 O 2 solution ph 11 buffered H 2 O 2 solution Part A: Enzyme Activity as a Function of Substrate Concentration An enzyme requires a substrate which it converts into product. Drawing from what you have learned about enzymes so far, develop a hypothesis regarding the effect of substrate (H 2 O 2 ) concentration on enzymatic reaction rate. Hypothesis: As substrate concentration increases, reaction rate will. Procedure 1. Obtain six test tubes and a test tube rack per group 2. Add 3 drops of blood and add to each of the six tubes 3. Using a dropper, add 3 drops of H 2 O 2 to test tube #1 4. After timing for 60 seconds, mark the maximum height of the bubble column with a Sharpie 5. Doing each tube one at a time, repeat steps 3 and 4 to the remaining five test tubes increasing by three drops the amount of H 2 O 2 in each test tube (i.e., test tube #2 receives 6 drops of H 2 O 2, test tube #3 receives 9 drops of H 2 O 2, etc.) Lake-Sumter State College, Leesburg Laboratory Manual for BSC 1010C 56

2 6. Upon completion, return to each test tube and measure in mm the distance from the bottom of the test tube to the height of the mark you made 7. Fill in Table 6.1 and plot the results as a bar chart in Fig. 6.1 Table 6.1 Reaction Rates for Catalase at Various Substrate Concentrations Test Tube Blood (# drops) H 2 O 2 (# drops) Height of Bubble Column (mm) Fig. 6.1 Reaction Rate of Catalase as a Function of Substrate Concentration Height of Bubble Column (mm) # of Drops of Substrate (H 2 O 2 ) Make a general statement regarding the effect of substrate concentration on enzymatic reaction rate: Lake-Sumter State College, Leesburg Laboratory Manual for BSC 1010C 57

3 Part B: Enzyme Activity as a Function of Temperature Temperature is a measure of the speed at which molecules are moving. As temperature increases, the molecular movement speed does so as well. Increasing temperature increases the probability and rate at which enzyme and substrate come together, thereby increasing the reaction rate. However, enzymes are subject to denaturation at excess temperatures. A denatured enzyme s active site conformation is changed not allowing the substrate to bind. The result is that at these temperatures, the reaction will decrease. An enzyme s optimum temperature is that point which has the greatest reaction rate but does not denature the enzyme. Drawing from what you have learned about enzymes so far, develop a hypothesis regarding the effect of temperature on enzymatic reaction rate. Hypothesis: As temperature increases, reaction rate will and then. Procedure 1. Obtain eight test tubes and a test tube rack per group 2. Using a pipette, measure 1 ml of H 2 O 2 into four of the eight test tubes 3. Using a dropper, add 3 drops of blood to each of the other four test tubes 4. Place one test tube each of H 2 O 2 and catalase into each of the water baths (make note of which tubes are yours for later retrieval). Leave the remaining pair of test tubes in the rack 5. Allow all tubes to acclimate for 15 minutes 6. After 15 minutes, proceeding one pair of tubes at a time, pour the H 2 O 2 into the catalase 7. After timing for 60 seconds, mark the maximum height of the bubble column with a Sharpie 8. Upon completion, return to each test tube and measure in mm the distance from the bottom of the test tube to the height of the mark you made 9. Fill in Table 6.2 and plot the results as a bar chart in Fig. 6.2 Table 6.2 Reaction Rates for Catalase at Various Temperatures Temperature ( C) Catalase (# drops) H 2 O 2 (ml) Cold 3 1 Room 3 1 Warm 3 1 Hot 3 1 Height of Bubble Column (mm) Lake-Sumter State College, Leesburg Laboratory Manual for BSC 1010C 58

4 Fig. 6.2 Reaction Rate of Catalase as a Function of Temperature Height of Bubble Column (mm) Temperature ( C) Make a general statement regarding the effect of temperature on enzymatic reaction rate: What is the optimum temperature for catalase? Lake-Sumter State College, Leesburg Laboratory Manual for BSC 1010C 59

5 Part C: Enzyme Activity as a Function of ph Another variable that can affect enzyme conformation and therefore activity levels is ph. Like with temperature, enzymes also have an optimum ph. Drawing from what you have learned about enzymes so far, develop a hypothesis regarding the effect of ph on enzymatic reaction rate. Hypothesis: As ph moves away from optimum, reaction rate will. Procedure 1. Obtain five test tubes and a test tube rack per group 2. Using a pipette, add 1 ml of buffered (3, 5, 7, 9, 11) H 2 O 2 to each of the five tubes 3. Using a dropper, add 3 drops of blood to test tube #1 4. After timing for 60 seconds, mark the maximum height of the bubble column with a Sharpie 5. Doing each tube one at a time, repeat steps 3-5 above to the remaining four test tubes 6. Upon completion, return to each test tube and measure in mm the distance from the bottom of the test tube to the height of the mark you made 7. Fill in Table 6.3 and plot the results as a bar chart in Fig. 6.3 Table 6.3 Reaction Rates for Catalase at Various Temperatures Test Tube ph Catalase (# drops) H 2 O 2 (ml) Height of Bubble Column (mm) Lake-Sumter State College, Leesburg Laboratory Manual for BSC 1010C 60

6 Fig. 6.3 Reaction Rate of Catalase as a Function of ph Height of Bubble Column (mm) ph Make a general statement regarding the effect of ph on enzymatic reaction rate: What is the optimum ph for catalase? Lake-Sumter State College, Leesburg Laboratory Manual for BSC 1010C 61

7 Practice Problems and Review Questions 1. What is meant by an organic catalyst? 2. List and describe the effect of the three major factors that cause changes in rate of enzymatic activity. 3. Why did certain temperatures and ph exhibit little or no activity at all? 4. During the reaction, you may have noticed a slight bit of heat given off. Explain the source of this heat. 5. What is the general equation for all enzymatic reactions? 6. Fill in these blanks The rate of enzymatic reaction is (directly / inversely) proportional to substrate concentration. At optimum, enzymatic reaction rate is (greatest / least). Lake-Sumter State College, Leesburg Laboratory Manual for BSC 1010C 62