Name: ____________________________________________ Period: ______ Date: _____________

 

General Biology

Effects of pH, temperature and Substrate on Enzyme Activity Lab

 

Introduction: Enzymes are proteins that speed up chemical reactions.  They do this by binding to the reactant(s), also referred to as the substrate, and forming an enzyme/substrate complex.  This complex places the reactant(s) in an orientation that facilitates bond breaking or bond formation thus lowering the activation energy needed to start the reaction.  The product(s) of the reaction form thousands of times more rapidly than they would without the enzyme.  When the reaction is complete, the enzyme itself can be reused on another molecule of substrate.

 

Each enzyme has a unique, three-dimensional shape that complements the substrate.  This relationship can be compared to how the shape of a key complements a lock.  As a result, enzymes are very specific and can only bond to one substrate.  Any environmental factor that denatures (alters) the shape of an enzyme such as high temperatures or extreme pH will prevent an enzyme from bonding to its substrate and catalyzing the reaction.

 

In addition to temperature and pH, the concentration of substrate available also affects enzyme function.  High concentrations of substrate result in high concentrations of product.  Most of the available enzymes will have substrates to bind with to facilitate product formation.  Low concentrations of substrate result in low concentrations of product.  Many of the available enzymes will not have substrates to bind with to facilitate product formation.  The impact of substrate concentration on enzyme function can also be seen as a reaction progresses.  When a reaction begins, large concentrations of substrate are present.  All of the enzymes will have substrates to bind with.  As the reaction continues, less and less substrate is present because much of it has been converted to product.  There is less of a chance of the enzyme binding to the substrate and therefore less likelihood of getting product.  

 

The enzyme you will be working with today is called catalase.  Catalase can be found in the cells of all living things.  It speeds up the following reaction by bonding to H2O2  (the substrate).

 

H2O2                             H2O    +     O2

 

  Hydrogen Peroxide                           Water              Oxygen

 

Hydrogen peroxide is produced from chemical reactions in your cells.  It is toxic in high concentrations.  Without the catalase enzyme, it will build up and kill the cell.

 

The amount of hydrogen peroxide broken down can be measured by the amount of oxygen released as a gas from the reaction.  In this lab, it will be measured by the amount of foam produced in the test tube. 

 

 

Purpose: The purpose of this lab is to investigate the effect of pH, temperature and substrate concentration on enzyme activity.

Materials:

6 test tubes                                                                  liver solution

1 M HCl                                                                      Boiled liver solution

1 M NaOH                                                                  Wax Crayon

pH paper                                                                     Hydrogen Peroxide

Graduated Cylinder                                                    stirring rod

Ruler                                                                          

 

Directions:

Part A

1)      Put on your goggles.  Leave them on for the entire lab.

2)      Obtain three test tubes labeled 1-3.  If they are not already clean, clean all test tubes with soap and water before you start.

3)      Using a graduated cylinder, measure 3 ml of water into tube one.  Measure 3 ml of liver solution in test tube 2.

4)      Scrub the graduated cylinder out with soap and water. 

5)      Use the clean graduated cylinder to measure 3 ml of boiled liver solution into test tube 3.

6)      Scrub the graduated cylinder out with soap and water.

7)      Use the graduated cylinder to add 5ml of 3% hydrogen peroxide to tube 1.  Measure the foam production in mm with a ruler immediately, and every 20 seconds until 80 seconds has passed.  Stir with a stirring rod in between measurements. (See figure 2.)

 

8)      Record the results in the data table 1 below.

9)      Rinse off the stirring rod.

10)  Add 5 ml of 3% hydrogen peroxide to the second tube.  Measure the foam production in mm with a ruler immediately, and every 20 seconds until 80 seconds has passed.  Stir with a stirring rod in between measurements. Record results in the table 1 below.  (WATCH OUT!!! YOU MAY WANT TO DO THIS ONE OVER THE SINK.)

11)  Record the results in the data table 1 below.

12)  Rinse off stirring rod.

13)  Repeat the same procedure for tube 3.

14)  Clean-up.  Rinse all test tube solutions down the sink with running water.  Wash all tubes, stirring rods, test tube racks, and graduated cylinders with soapy water.  (Anything that came into contact with liver must be washed.)  Wash off your lab station.  Dry everything with a paper towel.  Reline your plastic tray with a fresh paper towel.  Call your instructor over for an inspection. 

15)  Complete the analysis and conclusion of the lab.

 

 

 

Part B

16)  Put on your goggles.  Leave them on for the entire lab.

17)  Obtain three test tubes labeled 4-6.  If they are not already clean, clean all test tubes with soap and water before you start.

18)  Using a graduated cylinder, measure 3 ml of liver solution into each of the test tubes 4-6.

19)  Scrub the graduated cylinder out with soap and water. 

20)  Use a clean graduated cylinder to add 3 ml of HCl into tube # 5.  Rinse the graduated cylinder out well.

21)  Use the graduated cylinder to add 3 ml of NaOH into tube #6.  Rinse the graduated cylinder out well.

22)  Use the clean graduated cylinder to add 5ml of 0.3 % hydrogen peroxide to tube 4.  The 0.3% (dilute) hydrogen peroxide is on the front lab desk. Measure the foam production in mm with a ruler immediately, and every 20 seconds until 80 seconds has passed.  Stir with a stirring rod in between measurements. (See figure 2.)

 

 

23)  Record the results in the data table 1 below.

24)  Rinse off the stirring rod.

25)  Add 5 ml of 3% hydrogen peroxide to tube 5.  Measure the foam production in mm with a ruler immediately, and every 20 seconds until 80 seconds has passed.  Stir with a stirring rod in between measurements. Record results in the table 1 below. 

26)  Record the results in the data table 1 below.

27)  Rinse off the stirring rod.

28)  Repeat the same procedure for tube 6.

29)  Clean-up.  Rinse all test tube solutions down the sink with running water.  Wash all tubes, stirring rods, test tube racks, and graduated cylinders with soapy water.  (Anything that came into contact with liver must be washed.)  Wash off your lab station.  Dry everything with a paper towel.  Reline your plastic tray with a fresh paper towel.  Call your instructor over for an inspection. 

30)  Complete the analysis and conclusion of the lab.

 

 

Data Table 1

 

 

Time (seconds)

Foam Height (mm)

Tube 1 (Water and H2O2)

Tube 2 (Liver and 3% H2O2)

Tube 3 (Boiled Liver and

3 % H2O2)

Tube 4

( Liver and 0.3% dilute H2O2)

Tube 5 (Liver, 3% H2O2, and HCl)

pH = _____

Tube 6 (Liver, 3% H2O2, and NaOH)

pH = _____

O seconds

 

 

 

 

 

 

 

20 seconds

 

 

 

 

 

 

 

40 seconds

 

 

 

 

 

 

 

60 seconds

 

 

 

 

 

 

 

80 seconds

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

NAME: __________________________________________________ PERIOD: ________

 

 

Effects of pH, temperature and Substrate on Enzyme Activity Lab Questions

 

Analysis:

 

  1. Identify which test tube is the control group in this experiment.

 

 

 

 

 

  1. Identify which test tubes are the experimental groups in this experiment.

 

 

 

Conclusion:

 

1.  What is the name of the enzyme in this lab?  Where did we obtain this enzyme from?

 

 

 

 

2.  What was the substrate in the lab?

 

 

 

 

3.  In which tubes was the hydrogen peroxide broken down into water and oxygen?  How could you tell?

 

 

 

 

4.  Test tube #2 used 3% hydrogen peroxide and test tube #4 used dilute (0.3%) hydrogen peroxide. What affect does the concentration of substrate have on product formation? 

 

 

 

 

 

5.  Why did the foam production in tube 2 eventually slow down?

 

 

 

 

 

 

6.  How do enzymes lower the activation energy to speed up chemical reactions?

 

 

 

 

 

 

7.  What is the relationship between the shape of an enzyme and the shape of its substrate?

 

 

 

 

8.  Is HCl a strong or weak acid?  Is NaOH a strong or weak base?

 

 

9.  Compare test tubes #2 (normal enzyme) and #5 (enzyme with acid added). What affect do strong acids have on enzyme activity (enzyme activity increases, decrease or stays the same)?

 

 

 

 

 

 

10. Compare test tubes #2 (normal enzyme) and #6 (enzyme with base added). What affect do strong bases have on enzyme activity (enzyme activity increases, decrease or stays the same)?

 

 

 

 

 

11.  Compare test tubes #2 (normal enzyme) and #3(enzyme that was heated). What affect do high temperatures (boiling) have on enzyme activity (enzyme activity increases, decrease or stays the same)?

 

 

 

 

 

 

12.  Why do high temperatures and strong acids and bases interfere with enzyme function?