Chemistry 131 Laboratory

Experiment 11: Enzymatic Activity of Lactase

Instructors Note: Do "B" Ahead Of Time To Test Conditions

Look over the Introduction for this lab.
Complete the pre-lab questions for this lab.
Look over the INSTRUCTIONS for this lab.

Laboratory

GOAL

The purpose of this experiment is to explore the properties of the enzyme lactase. By the end of this experiment you should understand the biological function of enzymes are and why they are important in the body. You should also realize the importance of environmental conditions (pH, temperature, etc.) on the function of enzymes.

INTRODUCTION

In this laboratory you will explore enzyme catalysis. Enzymes are a class of protein molecules that act as biological catalysts. You may already know some properties of chemical catalysts. They increase the rate of a reaction, but are not used up in the reaction. Enzymes share these features with chemical catalysts, but also have some important differences. One of the most important differences is that enzymes are usually much more specific than chemical catalysts. Enzymes recognize particular reactant molecules, called substrates, and convert them into products. In contrast, chemical catalysts usually act on a broad range of reactants. In an enzymatic reaction, the enzyme “recognizes” the substrate by its shape and by the position of its hydrogen bonding sites and binds the substrate to itself at the active site. Once at the active site, the substrate rapidly undergoes the enzyme catalyzed reaction.

The enzyme you will be studying in this experiment is lactase, and the reaction it catalyzes is the hydrolysis of the disaccharide lactose into galactose and glucose. Humans require this enzyme for digestion of lactose found in milk and other dairy products. The medical condition known as lactose intolerance results when the body produces insufficient amounts of lactase to digest the lactose found in these foods. Lactose intolerance can cause intestinal discomfort and symptoms such as cramps, bloating and diarrhea. In the dairy case at the grocery store, you may have noticed 'lactose-free' milk. This type of milk has lactase enzyme added to it to hydrolyze the lactose before you drink it. Individuals with lactose intolerance can then enjoy the benefits of milk without the unpleasant side effects.

Although enzymes are very good at catalyzing reactions, they are also very sensitive to conditions in their environment. A small change in temperature or pH can have a dramatic influence on the rate of an enzyme catalyzed reaction. In your body, this is not a serious problem because the internal temperature and pH are carefully regulated. However, in a test tube the temperature or pH can be easily changed to observe the effect on reaction rate. Using systematic variation of reaction conditions, one can discover the details of how the enzyme functions. For certain enzymes, this information can be used to develop drugs that influence reactions in the body and benefit human health.

In this experiment, you will monitor the enzyme activity by determining how much glucose has formed. Because glucose is a product of lactose hydrolysis, measuring the amount of glucose gives a direct measurement of how much lactose has reacted. You will monitor the enzyme activity using test strips that measure the level of glucose in the reaction mixture. Because glucose is a product of lactose hydrolysis, measuring the amount of glucose gives a direct measurement of the extent of reaction. These are the same strips that diabetics use to monitor glucose levels with a home test kit. The presence of glucose in solution will change the test strip from yellow to a blue-green color. At this point you might be wondering "How do the test strips work? Glucose isn't green. Is it magic?" No, magic is not involved here, just chemistry. In fact, all test strips and test reagents that you might use for patient diagnosis in the hospital are based on chemical reactions. Here's how the glucose test strips work. First, the glucose is oxidized to form gluconic acid and hydrogen peroxide in a reaction catalyzed by a second enzyme called glucose oxidase. Then the hydrogen peroxide reacts with a dye, tetramethylbenzidine, and changes the dye from yellow to green. All of the detection reagents are impregnated into a pad on the surface of the strip, and the reactions take place inside the pad. By comparing the color of the strip to a standard color chart, the amount of glucose can be determined. The diagram below shows the reactions involve in glucose detection.

This laboratory is divided into four parts. In each part, you will be making systematic changes in the reaction conditions for lactase catalyzed hydrolysis of lactose. The conditions you will be changing are the temperature of the reaction, the pH of the reaction, the identity of the substrate and the amount of time the reaction is allowed to proceed. Your goal is to determine the influence of each variable on the enzymatic activity of lactase. By the end of the laboratory period, you should discover the optimal conditions for lactase activity.

Instructions

Throughout this experiment you will be performing enzyme assays. An assay is nothing more than a measurement of enzymatic activity, which is the amount of product produced in a given amount of time. For each part of the experiment, you will make an assay mixture by combining all of the components necessary to measure the enzyme activity except for the enzyme itself Then you will add the enzyme solution to start the reaction at a specific time and measure the amount of glucose produced after a certain amount of time has elapsed.

A STANDARD ASSAY MIXTURE for this experiment contains the components listed below. You will be making this solution several times for each part of the experiment.

1.0 mL of lactose stock solution (100 mg/mL)
1.0 mI, of pH 7.0 buffer
2.0 mL of distilled water

A) MAKING THE ENZYME SOLUTION

Put 25 drops of Lactaid® into 25 mL of distilled water. Mix the solution by inversion several times and store it on ice until needed.

B) LACTASE ACTIVITY OVER TIME

Make a standard assay mixture (Part A) in a small test tube using the recipe given above. Add 1.0 mL of the enzyme solution that you just made to the tube, mix by inversion and note the exact time. Measure the glucose concentration immediately and every thirty seconds for five minutes using glucose test strips. The procedure for using the test strip is to dip it into the assay solution for one second and then blot the side of the strip on a piece of tissue to remove excess solution. If you do not blot each strip, the glucose concentration will be abnormally high. Wait at least two minutes for the color to develop and compare you test strip to the color chart on the container. Record the concentration of glucose for each strip in your notebook.

C) LACTASE ACTIVITY AT VARIOUS TEMPERATURES

Set up five test tubes each containing the standard assay mixture given above. Put one in a boiling water bath, one in the 600 water bath, one in the 37° water bath, one at room temperature and one in an ice water bath, and wait a few minutes for the tubes to reach the appropriate temperatures. Add 1.0 mL of enzyme solution to the tube in the boiling water bath, mix by inversion and measure the glucose concentration after exactly five minutes of incubation at that temperature. Repeat this procedure using the standard assay mixture for the other four tubes until you have measured the enzyme activity at each temperature after exactly five minutes of incubation. For parts 'D' and 'E' you should share data with another group. Each group should perform either part 'D' or part 'E' and then exchange data with a nearby group that has done the other part of the experiment.

D) LACTASE ACTIVITY AT VARIOUS pH LEVELS

Fill five small test tubes with the solutions listed below. Notice that this makes five assay mixtures that are identical to the standard assay mixture except that each tube contains buffer of a different pH.

pH 2.0 assay mixture - 1.0 ml, lactose stock solution + 1.0 ml, pH 2.0 buffer + 2.0 mL distilled water
pH 4.0 assay mixture - 1.0 mL lactose stock solution + 1.0 mL pH 4.0 buffer + 2.0 m L distilled water
pH 6.0 assay mixture - 1.0 ml, lactose stock solution + 1.0 mL pH 6.0 buffer + 2.0 mL distilled water
pH 8.0 assay mixture - 1.0 mL lactose stock solution + 1.0 ml, pH 8.0 buffer + 2.0 mL distilled water
pH 10.0 assay mixture - 1.0 ml, lactose stock solution + 1.0 ml, pH 10.0 buffer + 2.0 mL distilled water

Add 1.0 mL of enzyme solution to the tube containing pH 2.0 assay mixture, mix by inversion and measure the glucose level after exactly five minutes. Repeat this procedure until you have measured the enzymatic activity at each pH after exactly five minutes.

E) SPECIFICITY OF LACTASE

Starch assay mixture - 1.0 mL starch stock solution + 1.0 mL pH 7.0 buffer + 2.0 mL distilled water
Maltose assay mixture - 1.0 ml, maltose stock solution + 1.0 ml, pH 7.0 buffer + 2.0 mL distilled water
Sucrose assay mixture - 1.0 mL sucrose stock solution + 1.0 mL pH 7.0 buffer + 2.0 mL distilled water
Fructose assay mixture - 1.0 mL fructose stock solution + 1.0 mL pH 7.0 buffer + 2.0 mL distilled water
Glucose assay mixture - 1.0 mL glucose stock solution + 1.0 ml, pH 7.0 buffer + 2.0 mL distilled water

F) SPECIFICITY OF LACTASE

Starch assay mixture - 1.0 mL starch stock solution + 1.0 mL pH 7.0 buffer + 2.0 mL distilled water
Maltose assay mixture - 1.0 ml, maltose stock solution + 1.0 ml, pH 7.0 buffer + 2.0 ml, distilled water
Sucrose assay mixture - 1.0 mL sucrose stock solution + 1.0 mL pH 7.0 buffer + 2.0 mL distilled water
Fructose assay mixture - 1.0 mL fructose stock solution + 1.0 mL pH 7.0 buffer + 2.0 ml, distilled water
Glucose assay mixture - 1.0 mL glucose stock solution + 1.0 ml, pH 7.0 buffer + 2.0 mL distilled water

Add 1.0 mL of enzyme solution to the tube containing the starch assay mixture, mix by inversion and measure the glucose level after exactly five minutes. Repeat this procedure with the other assay mixtures until you have measured the enzymatic activity for each one.

DATA ANALYSIS

For part 'B' draw a graph of glucose concentration vs. time. Draw a "best fit" straight line through the data points. Calculate the slope of this line, which is the rate of the enzymatic reaction.

Using data from 'C' and 'D', you should decide what temperature and pH give the highest level of lactase activity. How do your results compare to the conditions in the small intestine where lactase is present in the body? The instructions on the Lactaid® package say to put several drops of it into a quart of milk, shake and refrigerate for 24 hours. Why does lactase require such a long time to hydrolyze the lactose in the milk under these conditions?

Considering your data from part 'E' why is there such a high measurement in the glucose assay mixture? Is lactase really catalyzing any reaction in the glucose assay? What carbohydrates other than lactose, if any, can lactase enzyme convert to glucose?

Pre-lab Questions

Use Your Text Book As A Resoruce

1) Define the terms catalyst, assay, substrate and active site.

2) In addition to lactose, two other common disaccharides are maltose and sucrose. Draw the equations for the hydrolyisis of maltose and sucrose, showing the structures of all reactants and products. Name the product monosaccharides.

3) What is the temperature of a human body? At what temperature would you expect lactase to operate most efficiently? Why?

4.) What are the approximate temperatures of ice water, room temperature, and boiling water in degrees Celsius?

5) What is the slope of the line in the graph below. (Don't forget the units.) Remember the equation for the slope of a line is
slope = change along vertical axis/change along horizontal axis

Department of Chemistry

Capital University

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