Factors Affecting Enzyme Activity: Lactose Intolerance

B. Effect of pHFactors Affecting Enzyme ActivityMaterials:10 cuvettes, about 6 mL of the substrate solution (ONPG), about 3 mL of theenzyme solution (lactase), Buffer solutions: pH 4, pH 6, pH 7, pH 8, and either pH 9or 10, disposable pipettes (5 mL and 1 mL), ice, test tubesB.1 You must have a reference blank for each pH buffer. Start with the pH 4 buffer. To makeyour reference blank, place 3 mL of the buffer solution in a cuvette using a 5 mL disposablepipette. Add 0.5 mL of the substrate (ONPG) to the cuvette using a 1 mL disposable pipette.Add 0.5 mL of distilled water to the cuvette. Mix by inversion three times.B.2 Use the first reference blank to calibrate the spectrophotometer (reread the section on Howto Calibrate the Spectrophotometer. Once calibrated, don’t turn the dials or you will loseyour calibration.B.3 Make the sample solution at the same pH that you calibrated the spectrophotometer. Place3 mL of the buffer in an empty cuvette. Add 0.5 mL of the substrate (ONPG) to a cuvetteusing a 1 mL disposable pipette.Adding the enzyme solution:Add 0.5 mL of the stock lactase solution to the cuvette with the buffer and substrate andquickly mix by inversion three times. Start timing the solution. Wipe off any fingerprintsor spills on the outside of the cuvette and place it in the spectrophotometer. Make sure thespectrophotometer is set to read absorbance. At exactly 2 minutes, record the absorbancereading of this solution.B.4 Make a new reference blank with the next pH buffer using the procedure in B.1.Recalibrate the spectrophotometer with the new reference blank. Again, once calibrated,don’t turn the dials or you will lose your calibration.B.5 Make a new sample solution with the new pH buffer, following the procedure in B.3. Followthe Adding the enzyme solution procedure.B.6 Repeat until you have a reading for each pH buffer.B.7 Make a graph of absorbance on the y-axis versus pH on the x‐axis. Draw a smooth curvethrough your data points. (Optional): use Microsoft Excel to graph the data. A template willbe provided.