Factors Affecting Enzyme Activity: Lactose Intolerance

Factors Affecting Enzyme ActivityA.3 Your next 5 cuvettes will contain different enzyme concentrations, but each contains thesame amount of buffer and substrate. Place 3 mL of the pH 7 buffer in each of the fivecuvettes using a 5 mL disposable pipette. Add 0.5 mL of the substrate (ONPG) to each ofthe cuvettes using a 1 mL disposable pipette.A summary of the dilution process is shown on the following page.A.4 Cuvette #1 will contain the most dilute enzyme solution. In a separate test tube, add 1 mLof the stock lactase solution and 2 mL of distilled water. Mix by inversion three times.Adding the enzyme solution:Add 0.5 mL of this diluted enzyme solution to one of the cuvettes with the buffer andsubstrate and quickly mix by inversion three times. Start timing the solution. Wipe off anyfingerprints or spills on the outside of the cuvette and place it in the spectrophotometer.Make sure the spectrophotometer is set to read absorbance. At exactly 2 minutes, record theabsorbance reading of this solution.A.5 Cuvette #2 will contain the next dilute enzyme solution. Use the following dilution. In aseparate test tube, add 1 mL of the stock lactase solution and 1.5 mL of distilled water. Mixby inversion three times. Follow the Adding the enzyme solution procedure.A.6 Cuvette #3 will contain the next dilute enzyme solution. Use the following dilution. In aseparate test tube, add 1 mL of the stock lactase solution and 1 mL of distilled water. Mix byinversion three times. Follow the Adding the enzyme solution procedure.A.7 Cuvette #4 will contain the next dilute enzyme solution. Use the following dilution. In aseparate test tube, add 1 mL of the stock lactase solution and 0.5 mL of distilled water. Mixby inversion three times. Follow the Adding the enzyme solution procedure.A.8 Cuvette #5 will contain the most concentrated enzyme solution. No dilution is necessary.Add 0.5 mL of the stock lactase solution to the last cuvette with the buffer and substrateand quickly mix by inversion three times. Start timing the solution. Make the absorbancemeasurement as before.A.9 Make a graph of absorbance on the y-axis versus relative enzyme concentration onthe x‐axis. Using a ruler, draw a straight line through your data points. (Optional): useMicrosoft Excel to graph the data. A template will be provided.