lab 5

Protocol

LAB 5: FACTORS AFFECTING ENZYME ACTIVITY

Written by Michelle Furlong; modified by Jere Boudell

K N OW L E D G E OB J E C T I V E S

1. Understand enzyme function.

2. Understand basic protein structure.

3. Understand and describe the difference between positive and negative controls.

S K I L L S O B J E C T I V E S

1. Practice identifying the components of scientific studies.

2. Practice writing hypotheses and predictions.

3. Using positive and negative controls to identify the potential outcomes of a test or experiment.

B A C K GR OU N D

An enzyme is a protein molecule that serves as a biological catalyst or a substance that can increase the rate of a reaction without changing its own structure. Enzymes catalyze reactions that occur inside living things (i.e., metabolic reactions). They work by binding to a substrate (reactant) at their active site to form an enzyme-substrate complex, and converting the substrate into product(s) of the chemical reaction. Once the enzyme converts the substrate it releases the product(s) and leaves the reaction in the same exact form it was introduced. Because enzymes are not altered during the reactions, they can be used repeatedly.

Enzyme + Substrate (enzyme-substrate complex) Enzyme + Product

Enzymes usually catalyze only specific reactions. For example, the enzyme amylase only catalyzes the conversion of starch to maltose. It does not catalyze any other biological reactions.

Like all proteins, enzymes are made up of a chain of covalently bonded amino acids (primary structure) that bend, turn and fold to form secondary, tertiary and quaternary structures, which make up its 3D shape. The shapes of enzymes are the result of weak bonds (i.e., hydrogen bonds) that occur between non-adjacent amino acids. This 3D shape allows them to properly catalyze reactions (i.e., function).

Enzymes function at a precise temperature and pH range. For instance, most enzymes found in the human body function most efficiently at 37 ºC (98.6 ºF). When an enzyme is outside its optimal temperature and pH range, it cannot function properly. The enzyme works more slowly or sometimes does not work at all.

Enzyme denaturation occurs when it is subjected to excessive heat or extremely high or low pH (denaturing conditions). When an enzyme is denatured, it loses its quaternary, tertiary and secondary structure and becomes a chain of amino acids linked by peptide bonds (or covalent bonds that occur between adjacent amino acids). This primary structure usually stays the same, because the peptide bonds are too strong to break. However, enzymes that have lost their 3D shape no longer function.

Protocol

In this experiment, you will be testing the enzyme catecholase which is an enzyme used by plants to convert catechol, a colorless and harmless liquid into benzoquinone, a brownish liquid that kills bacteria. This reaction can only occur in the presence of large amounts of oxygen, such as when a plant is cut, and its cells are exposed to the air. Thus, the plant produces a chemical to kill bacteria whenever its skin is broken. This reaction is what causes fruit to brown soon after being cut. So, the fruit is not rotting; this is the fruit attempting to defend itself from bacteria.

E X E R C I S E 5 . 1 – E N Z YM E A C T I V I T Y

In this exercise, you will be testing the enzyme catecholase which is responsible for the browning of cut apples.

Catecholase + Catechol catecholase-catechol complex) Catecholase + Benzoquinone

Enzyme + Substrate (enzyme-substrate complex) Enzyme + Product

P R E P A R A T I ON O F FO OD S

For each of these experiments, you will need to cut “fresh” slices of apple. DO NOT cut all the apples at once as they will begin to brown. Cut them as needed for each experiment.

E N ZY ME A C T I V I T Y P A R T A

The substrates for the enzyme catecholase are catechol and oxygen. For this part of the exercise, we will exclude oxygen from the reaction to prove that it is needed.

D I R E C T I O N S

1. Cut two fresh slices of apple. 2. Fill a bowl with water. Place one slice of apple into the water and set one slice aside on a plate

(make sure the water covers the apple completely in the bowl). 3. Observe the slices every 30 minutes for 2 hours and record your results

E N ZY ME A C T I V I T Y P A R T B

In this exercise, you will test the effects of extreme heat (100 ˚C) and low pH (very acidic) on enzyme activity (i.e., function). To test the pH of common household items, you will be using pH paper or creating pH sensitive paper by microwaving red cabbage leaves.

This experiment has a positive control and a negative control for comparison. The negative control lacks oxygen; therefore, there should be no browning. It is considered a negative control because it will test negative for enzyme activity (catechol will be present). The positive control contains benzoquinone; therefore, there should be browning. It is considered a positive control because it will test positive for full potential enzyme activity (i.e., the enzyme functioned).

2 – Lab 5: Factors Affecting Enzyme Activity

Protocol

D I R E C T I O N S

Enzyme Denaturation by pH

1. If you were unable to purchase pH paper, you can make pH paper to be able to identify common acids and bases around the house. (If you have pH paper, skip to step 8)

2. Blend or grate ½ of a red cabbage head. If you don’t have a blender or grater, then chop up cabbage using a knife.

3. Place the cabbage shreds in a bowl and microwave until steaming hot (Microwave times will vary between 1 and 3 minutes). If you don’t have a microwave, you can cook the cabbage on the stove. The goal is to cook the cabbage enough that the plant cells burst and release the pigment inside them.

4. Pour the cabbage juice through a mesh strainer or colander and capture the liquid. Throw away the cabbage shreds.

5. Pour the strained cabbage juice through a coffee filter and capture the fluid. 6. Cut a coffee filter into 1-inch squares and soak them in the cabbage juice for 5 minutes. Allow

them to dry. 7. The paper will change colors depending on the pH of the liquid placed on it. Neutral pH (7) will

not change the color, though it will make the color seem darker due to wetness. Weak bases (9- 10) will give a blue color and strong bases (12-14) will give a green color. Weak acids (3-5) will give a violet or red color and strong acids (1-2) will give a pink color.

8. Place a drop of water, a drop of lemon juice, a drop of white vinegar, and a drop of ammonia onto a separate pH test paper. Determine the pH of each solution.

9. Cut 4 fresh slices of apple. Soak one slice of apple in each of the four solutions for 5 minutes. 10. Pull the slices out of the solutions and set them on a plate. Observe the slices every 30 minutes

for 2 hours and record your results.

Enzyme Denaturation by Temperature

1. Cut one fresh slice of apple and boil for10 minutes. 2. Cut another fresh slice of apple (room temperature) and place on a plate with boiled apple slice. 3. Put a fresh apple slice in the refrigerator and a fresh apple slice in the freezer. 4. Observe the slices every 30 minutes for 2 hours and record your results.

3 – Lab 5: Factors Affecting Enzyme Activity