Lactase is an enzyme that breaks down lactose sugar to glucose and galactose in the small intestine, kidneys and liver. A lactase enzyme assay is a standard first year biochemistry lab experiment. Part of this lab involves measuring the efficiency of lactase at various pH levels to determine its ideal pH. You can accomplish this by mixing different buffer solutions with a measured amount of lactase and its substrate, then recording the efficiency of lactase in a spectrophotometer.
Things You'll Need
Felt-tip pen
7 spectrophotometer cuvettes
3.5 ml of ONPG (lactose substitute)
4.5 ml of each buffer solution: pH 2, 4, 6, 7, 8, 10, 12
Pipettes
Spectrophotometer
Ballpoint pen
Lab book
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Instructions
Measuring pH of lactose solution
1
Do not mark the window of the cuvette. This spoils the experiment.
Label seven cuvettes by number with the felt-tip pen.
2
ONPG is a lactose substitute. It is the subtrate for this enzymatic reaction.
Add 0.5 ml of ONPG to each cuvette using a clean, dry pipette.
3
Take careful notes in your lab book.
Add 4.5 ml of each buffer solution into separate cuvettes. For example: add the pH 2 buffer solution into cuvette #1. Add the pH 4 buffer solution to cuvette #2. Continue in this manner until all the cuvettes are filled. Use a different pipette for each buffer solution to prevent contamination. Clearly mark the cuvette number and the pH of the buffer solution it contains in your lab book.
4
Move as quickly as possible.
Using a clean-dry pipette, quickly add 0.4 ml of the lactase enzyme to each cuvette.
5
Work quickly. The reaction has already started.
Place the cuvettes in the spectrophotometer, close the lid and run the pH assay. The results of the assay will tell you the ideal pH for a lactase reaction.