Factors Affecting Enzyme Activity: Lactose Intolerance

C. Effect of Substrate ConcentrationFactors Affecting Enzyme ActivityMaterials:7 cuvettes, about 7 mL of the substrate solution (ONPG), about 4 mL of the enzymesolution (lactase), about 20 mL of pH 7 buffer solution, disposable pipettes (5 mLand 1 mL), ice, test tubesC.1 To make your reference blank, place 3 mL of the pH 7 buffer in a cuvette using a 5 mLdisposable pipette. Add 0.5 mL of the enzyme (lactase) solution to the cuvette using a 1 mLdisposable pipette. Add 0.5 mL of distilled water to the cuvette. Mix by inversion threetimes.C.2 Use the reference blank to calibrate the spectrophotometer (reread the section on How toCalibrate the Spectrophotometer. Once calibrated, don’t turn the dials or you will lose yourcalibration.C.3 Your next 4 cuvettes will contain different substrate concentrations, but each contains thesame amount of buffer and enzyme. Place 3 mL of the pH 7 buffer in each of four cuvettesusing a 5 mL disposable pipette. Add 0.5 mL of the enzyme (lactase) to each of the cuvettesusing a 1 mL disposable pipette.A summary of the dilution process is shown on page 10.C.4 Cuvette #1 will contain the most dilute substrate solution. In a separate test tube, add 1 mLof the stock substrate (ONPG) solution and 2 mL of distilled water. Mix by inversion threetimes.Adding the substrate solution:Add 0.5 mL of this diluted substrate solution to one of the cuvettes with the buffer andenzyme 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.C.5 Cuvette #2 will contain the next dilute substrate solution. Use the following dilution. In aseparate test tube, add 1 mL of the stock ONPG solution and 1 mL of distilled water. Mixby inversion three times. Follow the Adding the substrate solution procedure.C.6 Cuvette #3 will contain the next dilute substrate solution. Use the following dilution. In aseparate test tube, add 1 mL of the stock ONPG solution and 0.5 mL of distilled water. Mixby inversion three times. Follow the Adding the substrate solution procedure.C.7 Cuvette #4 will contain the stock substrate solution; no dilution is necessary. Add 0.5 mLof the stock ONPG solution to the cuvette with the buffer and enzyme and quickly mix byinversion three times. Make the absorbance measurement as before.C.8 Cuvettes #5, and #6 contain different amounts of buffer and substrate than your first fourcuvettes! Read carefully how to make these last two samples.In cuvette #5, place 2.5 mL of the pH 7 buffer in the cuvette using a 5 mL disposable pipette.Add 0.5 mL of the enzyme (lactase) to the cuvette using a 1 mL disposable pipette.Add 1.0 mL of the stock ONPG solution to the cuvette with the buffer and enzyme and