Safety Precautions

1. Tie hair back. Wear proper clothing, like closed toed shoes.

2. Avoid all contact with the hot plate. Use beaker tongs when necessary.

3. Wear goggles when working to avoid any contact with substances.

4. Don't smell the substances, and be careful not to inhale them.

Purpose

The purpose of this lab is to further understand enzymes, their reactions, and their substrates. In particular, this lab explores the properties of the enzyme lactase, its product, glucose, and the difference in reaction between sucrose and lactose.

Introduction

Enzymes act as biological catalysts. However, unlike chemical catalysts, they are more specific. In an enzymatic reaction, an enzyme recognizes the shape of a substrate, and if the structure and shape is right, the substrate bonds in the active site.

We will be working with lactase, and its native substrate lactose. Lactose is made out of galactose and glucose. Glucose is a product of lactose hydrolysis which is the chemical breakdown of a compound due to reaction with water. By measuring the amount of glucose, we can measure how much lactose has reacted by putting glucose test strips into each solutions. If the strip turns from blue to green, we know that there is glucose present in the reaction.

The other potential substrate we will be working on is sucrose. The monosaccharides compose sucrose are glucose and fructose.

In this experiment, we evaluated if there was glucose in five solutions: (1) milk and enzyme solution (2) milk and water solution, (3) milk and denatured enzyme solution, (4) sucrose and enzyme solution, and (5) sucrose and water solution.

Procedure and Materials

Our lab procedure was the following:

Solution Preparation:

Make Enzyme Solution:

Add 1 lactase tablet to 200 mL of water. Stir until dissolved using stirring rod.

Gather Skim Milk.

Make Sucrose Solution:

• Add 5 grams of sugar to 100 mL of water. Stir until dissolved using stirring rod.

Make Denatured Enzyme Solution:

• Put 20 mL of Enzyme Solution into a test tube.
• Add 200 mL of water to a 400 mL beaker.
• Place the test tube with Enzyme Solution into the beaker with water, being careful to rest tube on side of beaker.
• Boil the water in the beaker for 30 minutes.
• Let solution cool to room temperature.

Lab Procedure:

Gather materials. (See Solution Preparation)

Label the test tubes with the following labels using tape and sharpies:

• A. Test tube with skim milk and enzyme solution.
• B. Test tube with skim milk and water.
• C. Test tube with skim milk and denatured enzyme solution.
• D. Test tube with sucrose solution and enzyme solution.
• E. Test tube with sucrose solution and water.

In test tube A, add 2 mL of skim milk and 1 mL of enzyme solution.

Time for 2 minutes and test for glucose with a glucose test strip. Record results in the Data Table. If there was glucose present, mark a + in the table. If not, mark -.

In test tube B, add 2 mL of skim milk and 1 mL of water.

Time for 2 minutes and test for glucose with the glucose test strip. Record results in the Data Table. If there was glucose present, mark a + in the table. If not, mark -.

In test tube C, add 2 mL of skim milk and 1 mL of denatured enzyme solution.

Time for 2 minutes and test for glucose with the glucose test strip. Record results in the Data Table. If there was glucose present, mark a + in the table. If not, mark -.

In test tube D, add 2 mL of the sucrose solution and 1 mL of enzyme solution.

Time for 2 minutes and test for glucose with the glucose test strip. Record results in the Data Table. If there was glucose present, mark a + in the table. If not, mark -.

In test tube E, add 2 mL of sucrose solution and 1 mL of water.

Time for 2 minutes and test for glucose with the glucose test strip. Record results in the Data Table. If there was glucose present, mark a + in the table. If not, mark -.

Clean up your lab station. All solutions can be poured down the sink in this lab. The used glucose test strips should be discarded in the trash. Wash out all glassware at the sink using soap and water. If your lab table is wet, please take a sponge and wipe down the table.

Observations and Data

In this lab, we looked for the presence of glucose by checking the color of our glucose test strips before and after we put them into the different test tubes. If the glucose test strip turned from blue to green, glucose was part of the reaction. However, for all but one of our solutions, no glucose was present.

Conclusions and Analysis

In this experiment, the only solution that contained glucose after the reaction was the milk and enzyme solution. All of the other solutions contained no glucose. Throughout this section, we will explain why this happened.

In a lactose and lactase reaction, lactose, the substrate, fits into lactase's (the enzyme) active site. This reaction is a hydrolysis reaction, which means it requires water to produce products. When the substrate lactose binds into lactase's active site, it forms the enzyme substrate complex. As the enzyme works on the substrate, it breaks down lactose into galactose and glucose, the new products. This is the enzyme product complex. Once transformed from substrate to products, the products leave the active site of the enzyme as galactose and glucose.

In this experiment, we compared the reactions of lactase with both lactose and sucrose. According to our pre-lab, sucrose and lactose are both disaccharides. They both have the same chemical formula, but sucrose is composed of glucose and fructose, rather than galactose and glucose. However, in our experiment, we noticed that whenever a solution contained sucrose, it did not produce glucose, showing us that the enzyme did not react to it. This is because the active site of lactase does not match the shape of sucrose. The structure is too different to fit in the active site, and therefore, products cannot be produced. Because of their structures, lactose and sucrose, which seemed so similar before, are actually quite different.

For solution C, we combined denatured enzyme solution and skim milk. Denatured means damaged. To make this "damaged" enzyme solution, we had to boil the enzyme. When boiling the enzyme, the high temperatures altered the shape of the enzyme and active site. Basically, boiling the enzyme makes it useless to substrates like lactose that previously fit in the active site. Because of this reaction, the denatured enzyme and skim milk solution tested negative for glucose.

In humans, lactase works best in the stomach -- an area of the body with a high pH/acidity. We can call this stomach pH the "optimum pH" of the enzyme. (In other words, the enzyme lactase works best when the acidity is just like it is in the stomach. Therefore, lowering the pH of the enzyme solution would have a negative effect on the reaction. It would decrease rate of reaction, and if it got too low, the reaction would not be possible.

As mentioned before, the lactase enzyme reaction is a hydrolysis reaction. Hydrolysis is a chemical breakdown of a compound due to a reaction with water. When water is added to lactose in lactase, it is able to break down the lactose into galactose and glucose.

This lab was very helpful in further explaining enzymes, substrates, and products.