BIOLOGY SEMESTER ONE

LAB: ENZYME ACTIVITY

LAB:ENZYME ACTIVITY ON LACTOSE

Lab format: this lab is delivered though a lab kit

Relationship to theory: In the textbook (Reece et al., 9th ed.), this lab is related to the following unit:

Learning Objectives

At the end of this laboratory, you should be able to:

  1. Define enzyme and describe enzyme activity in cells
  2. Discuss the effects of various environmental conditions on the rate of activity of enzymes.
  3. Acknowledge enzyme specificity and relate it to cell function.
  4. Practice techniques in performing assays.
  5. Produce graphical representations of empirical data and interpret the results.

Introduction

Enzymes are globular proteins that regulate nearly all metabolic processes within living organisms by controlling the point at which catalysis takes place. Enzymes work as biological catalyst, by lowering the activation energy for a reaction, thus increasing the rate of a reaction. For example, carbonic anhydrase can Increase the reaction rate of carbon dioxide binding to water to produce carbonic acid by 10 million times. Enzymes are crucial, as we could not rely on physical catalysts such as heat and pressure to work within our cells (they would cause too much damage in such a delicate system). Enzymes are usually named for the molecule they work on; the nomenclature then ends in “ase.” For example lactase is the enzyme that is responsible for the breakdown, or hydrolysis, of lactose (the sugar found in milk).

Enzymes tend to be very specific as to which reactions they catalyze. The shape of the enzyme, its charge and it hydrophilic/hydrophobic characteristics all contribute to its exclusiveness. Enzymes bind to the substrate, or molecule on which they will act, at the active site. This site is a groove in the surface of the enzyme, somewhat like the cleft in a plum. At the active site the enzyme and substrate adjust their shapes somewhat to create what is known as an “induced fit”; the molecules are now in extremely tight contact. This closeness allows the amino acid side groups of the enzyme to cause stress and distortion on the substrate molecule’s bonds, and it is thought that this is how the enzyme is able to lower the activation energy of certain reactions. Once the reaction takes place, the enzyme releases the resulting products, and, as it has not been altered by the encounter, it can attach to a new substrate to catalyze another reaction.

Enzymes, like all proteins, are made of long linear chains of amino acids that fold into tertiary, three dimensional shapes. The arrangement of the amino acids creates the bonds that cause the folding, and therefore contribute to the distinctive structure of each enzyme. Like all proteins, most enzymes can be denatured (unfolded and deactivated) by heat or chemical interactions. Depending on the enzyme, this process may or may not be reversible. Lactase, the enzyme you will be investigating in this lab, does not denature in high heat.

Enzyme activity can be affected by factors such as substrate concentration, temperature, and the chemical environment (for example pH). Most enzymes will have optimal working conditions for all of these factors; we will investigate some of these parameters in this lab.

Additionally, enzyme activity can be affected by other molecules called inhibitors (which decrease activity) or activators (increase activity). Aspirin is a common enzyme inhibitor, and inhibits COX-1 and COX-2 enzymes that produce prostaglandin. Prostaglandin is responsible for carrying messages of inflammation and pain to and from the brain. Aspirin acts to interrupt the enzymes from sending the message, thus blocking the sensation.

Enzymes have a variety of functions: they serve living orgasms in all aspects of cell metabolism, generate light in fireflies, and assist viruses in infecting cells. Some of the more common enzymes are those that function to break down food into molecules so that they are readily absorbed by an organism’s digestive system. Amylases breaks down starch, but the resultingmolecules are still too large to be absorbed. The molecules are then worked on by other enzymes, further down the intestinal tract, that break starch into simple sugar (glucose) so it can be readily absorbed. Organisms with specialized diets tend to have specialized digestive enzymes. This is also why some foods are toxic to some species and not others.

Enzyme activity plays a crucial role in homeostasis. A single mutation in an amino acid resulting in an alteration in enzyme production can lead to severe results such as heredity cancer or mental retardation. However, it can also cause much less serious consequences, such as the inability to breakdown alcohol or dairy sugars.

Throughout this experiment you will be performing enzyme assays. An assay is a measurement of enzymatic activity. This can be quantified as the amount of product produced in a given amount of time. Lactase is an enzyme involved in the hydrolysis of lactose, the sugar found in milk. Hydrolysis occurs when the disaccharide (two-sugar) lactose is split into two smaller glucose molecules and water. Lactase is found predominantly in the small intestine in humans, and a deficiency can cause lactose intolerance, resulting in gastrointestinal upset as the undigested sugars in the intestine ferment and produce gasses. As most people reach adulthood, lactase production decreases, but there are studies that show there are regional differences among populations in the decline of lactase production during a lifetime.

Figure 3.1: Lactose Molecule

Image From:

equipment

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BIOLOGY SEMESTER ONE

LAB: ENZYME ACTIVITY

  • Spot plate
  • Marker
  • Toothpick for mixing
  • Forceps for handling the glucose test paper
  • Cow’s Milk (whole, skim, 1% or 2%)
  • Pipettes
  • Lactaid® (lactase source) liquid form or Extra strength caplets
  • Glucose test strips (such as Bayer Diastix®)
  • Glucose tablet or corn syrup
  • white table sugar
  • Plastic bottle with lid
  • Ice cubes
  • Thermometer
  • Stir stick
  • Stop watch
  • Camera

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BIOLOGY SEMESTER ONE

LAB: ENZYME ACTIVITY

Procedure 1: Testing for lactase activity
  1. Prepare the enzyme solution in a 100mlbeakerby mixing 25 drops of Lactaid® into 25 ml of distilled water. Mix the solution by inverting it several times and then store it on ice until needed. If you are using the caplet form, grind two caplets to a fine powder using a mortar and pestle or by placing the caplets in a clean Ziploc bag and crushing them with a rolling pin or hammer. They should be a fine powder. Place the powder in a 100 ml beaker and add a few drops of distilled water. Stir and continue adding small amounts of water until you have a smooth paste. Top up to the 50ml mark and store on ice until needed.
  2. Prepare the glucose solution by crushing a 4g glucose tablet; mix with 200 ml of water, and stir until dissolved. Alternatively, make a solution using 2 ml of corn syrup to 100 ml of distilled water. Stir until dissolved.
  3. Prepare the sucrose solution by mixing 100ml of water with 2g of white table sugar.
  4. Use a marker to number 4 depressions on the spot plate.
  5. Using different pipettes for each, add the following solutions:

Spot 1: 2 drops of glucose solution;
Spot 2: 2 drops of milk;
Spot 3: 2 drops of lactase;
Spot 4: 1 drop of lactase plus 2 drops of milk (mix with toothpick).

  1. After three minutes, test each of the spots for the presence of glucose by testing with the glucose test strip. When using the strip, dip it in the solution and then tap the excess off onto a piece of tissue (this ensures better accuracy in the results).
  2. After 30 seconds, note the concentration of glucose by comparing the strip with the colour chart provided.
  3. Record the data in table 1 and complete the table, with your brief interpretation of the results..

TABLE 1: RESULTS OF TEST FOR GLUCOSE

Spot / Contents / Testcolor / Interpretation
1 / Glucose
2 / Milk
3 / Lactase
4 / Lactase + Milk

Questions

  1. What products would result after this hydrolysis?How did you know this? (3 marks)
  2. Does milk contain glucose?What is your proof? (2 marks)
  3. Does milk combined with lactase contain glucose? What is your proof? (2 marks)
  4. Why is it necessary to use cow’s milk in this experiment? Would we get similar results with other types (such as goat or soy)? Why or Why not? (4 marks)

Procedure 2: Lactase activity over time

  1. Pipette 10ml of milk into a test tube.
  2. Add 3.0ml of enzyme solution (prepared in previous procedure). Take note of the time.
  3. Immediately test the glucose concentration of the mixture, and then take glucose readings every minute for a total of 10 minutes using the glucose test strips. Remember to tap the strip on a piece of tissue to remove excess solution.Do not wait until the end to read the sticks as they are time sensitive. Be sure to check the glucose strips 30 seconds after they have been dipped.
  4. Record your data in the following table.

Table 3.2: Results of Glucose concentration over time

Time / Glucose Concentration / Time / Glucose Concentration
1 min / 6 min
2 min / 7 min
3 min / 8 min
4 min / 9 min
5min / 10 min

Questions

  1. Using Excel, create a graph of glucose concentration vs. time. Calculate the slope of this line, which represents the rate of the enzymatic reaction. (5 marks)
  2. Predict what the extension of this graph would look like. Would glucose concentration increase infinitely with infinite time? Why or why not? (4 marks)

Procedure 3: Lactase Activity at Various Temperatures

  1. Prepare five different temperature water baths for your test tubes as outlines below. You may use a heat safe container like a beaker, mug, or Pyrex glass to rest your test tubes in. Use a thermometer to ensure you match temperatures as closely as possible. Be sure the water level is low enough to accommodate the test tube without flooding it, and that your container is narrow enough to support the test tube vertically. Work quickly in order to maintain the temperatures for the duration of the test.
  2. Bath 1: Ice bath- fill a cup with ice and then add water to fill in the spaces.
  3. Bath 2: 37°C- adjust tap water until it measures 37°C and make a water bath for test tube 2.
  4. Bath 3:50°C-adjust tap water until is measures 50°C and make a water bath for test tube 3.
  5. Bath 4: Room temperature-adjust tap water to 21°C and create a water bath for test tube 4.
  6. Bath 5: Boiling water bath- heat water in a kettle or microwave until it reaches the boiling point. It should be approximately 100°C. Make a water bath for test tube 5.
  7. Place each of the test tubes in their corresponding water bath and measure 10ml of milk into each one. Wait a few minutes for the milk temperature to adjust to its surrounding environment. Recheck the bath temperatures at this time and carefully adjust as necessary (to within 1 or 2°C).
  8. Pipette 3.0 ml of enzyme solution into each of the five test tubes, stirring gently to ensure a homogenous mixture. Take note of the time.
  9. Incubate the mixtures at their respective temperatures for five minutes.
  10. Take concentration readings of each of the test tubes using the glucose test strips.

Table 3.3: Glucose concentration at various temperatures

Initial Water Bath Temperature
(record actual temperature in °C) / Glucose Concentration After 5 minutes / Final Temperature (°C)
1.
2.
3.
4.
5.

Questions

  1. Using a spreadsheet program (e.g. Microsoft Excel), graph glucose concentration vs. temperature, including all appropriate labels. (5 marks)
  2. At what temperaturedid you observe the highest level of lactase activity? (1 mark)
  3. Discuss how yourresults compare to the conditions in the small intestine wherelactase is present in the body. (2 marks)
  4. The liquid Lactaid®package instructs users to put several drops into 2 liters of milk,shake, and refrigerate for 24 hours. Explain, in terms of your findings, why so much time is needed for the milk to be acted on under these conditions. (2 marks)
  5. It was stated that lactose does not denature when exposed to heat. Identify two enzymes that do denature in response to increasing temperature. At what temperature do these two enzymes respectively breakdown? What are their optimal temperatures? How do their optimal temperatures and denaturing temperatures relate to their function? (6 marks)

Bibliography

Capital University. (1998). Chemistry 131 Laboratory: Enzymatic Activity of Lactase. Retrieved 10 19, 2009, from Department of Chemistry:

Crumlish, J. (2008, 04 20). Activities Exchange. Retrieved 01 02, 2010, from National Health Museum:

Tortora, G. a. (1993). Principles of Anatomy and Physiology. New York: Harper Collins College Publishers.


NANSLO Biology Core Units and Laboratory Experiments
by the North American Network of Science Labs Online,
a collaboration between WICHE, CCCS, and BCcampus
is licensed under a Creative Commons Attribution 3.0UnportedLicense;
based on a work at rwsl.nic.bc.ca.
Funded by a grant from EDUCAUSEthrough the Next Generation Learning Challenges.

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