11 Lab Protocol: Enzymes

Last week you tested several milk products using three reagents to test for the presence of proteins, carbohydrates, and reducing sugars. This week, you will continue testing milk products and exploring the activity of enzymes on proteins in nonfat milk.

As you’ve learned in lecture, enzymes catalyze reactions and the rate of a reaction relies on physical factors such as temperature and pH. You will be working with the enzyme trypsin. Trypsin is a digestive enzyme that plays a crucial role in protein digestion. In humans, it is released from the small intestine can break down large protein molecules into smaller ones to be further broken down by other enzymes. In powedered milk, trypsin will break down the protein casein into smaller soluble peptides and amino acids.

Test 1: Control experiment to measure the rate of reaction of trypsin on milk protein at 40 degrees C:

1. Read all the steps in this procedure and write down your hypothesis for the experiment you will test in your data sheet.
2. Obtain 3 test tubes and label one with a C for control, one with a T for trypsin, and one with an E for experiment solution.
3. Use a transfer pipette and add 1 mL of ph 7 buffer to each of the control and experiment test tubes.
4. Next, add 5 mL of nonfat milk to each of the control and experiement test tubes.
5. Use a transfer pipette and add 3 mL of trypsin to the trypsin test tube.
6. Place all three test tubes in a 40 C water bath for 5 minutes.
7. Now you will run your experiment. Pour the trypsin into the tube labeled E and set a timer right away to test how long it will take for trypsin to digest casein completely. When casein is digested, the milk solution will turn from opaque to a translucent. You will also see a white sediment at the bottom of the test tube.
8. Record your results in your data sheet.

Test 2: Designing an experiment to determine effect of temperature on enzyme activity:

1. In this experiment, you will repeat the procedures from Test 1, which was your control experiment at different temperatures to measure the effect of temperature on the reaction rate.
2. Discuss with your group how you will run the experiment at 3 additional temperatures: 0 C, 80 C, and 100 C.
3. Run your experiments and record your results in your Student Data Sheet.

Test 3: Designing an experiment to determine the effect of pH on enzyme activity.

1. In this experiment, you will repeat procedures from Test 1, which was your control experiment, at different pH levels.
2. Discuss with your group how you will run the experiement at 3 additional pH values. There are 3 different buffer solutions for you to use.
3. Run your experiments and record your results in your Student Data Sheet.