Enzymes
Objectives
1)To understand the importance of the relationship of structure to enzyme function.

2)To be familiar with how enzymatic reactions are influenced by changes in:

a.Enzyme concentration

b.Substrate concentration

c.pH

d.Ionic concentration

e.Temperature

f.Inhibitor (CuSO4)

3) To identify the independent and dependent variables in each of the experiments A-F.

Introduction

In addition to your textbook, below are some web resources that will add more background information about enzymes:

http://ntri.tamuk.edu/cell/enzyme2.html

http://www.worthington-biochem.com/introBiochem/introEnzymes.html

 

In each individual cell of a human there are many chemical reactions taking place, performing the necessary functions for being a large, complex, multicellular organism. This is relatively easy to understand. How do these reactions occur? This is not so easy to understand. Chemical reactions involve the breaking and reforming of chemical bonds between molecules (substrate(s) of the reaction), which are transformed into different molecules (product(s) of the reaction). Chemical reactions can occur spontaneously (without added energy or intervention), and indeed many of the chemical reactions necessary for life processes are spontaneous; some however, are not. Metabolic pathways are processes, which involve many chemical reactions that occur in a specific order.

For example, to get energy out of a molecule of glucose, a series of reactions must take place in a specific order to break the bonds between the carbons of the glucose molecule. In addition, you have to rely on a series of chemical reactions that break down stored glycogen into glucose molecules to have glucose molecules in the first place. If you had to rely on these reactions to take place spontaneously, you would wait a very long time -- you wouldn't be here! Enzymes catalyze chemical reactions so that they occur in a timely and sequential manner to produce a product.

Enzymes are biological catalysts. They help to increase the rate of chemical reactions. Enzymes are most often proteins and their three-dimensional shape is important to their catalytic activity. Because of their 3-D shape, enzymes are highly specific for the reactions that they catalyze. In other words, they are highly specific for the substrates that they will act upon. So any one "function", such as getting energy from a glucose molecule, actually involves many reactions, each with a specific enzyme.

Enzyme activity is influenced by many factors. You will be examining some of the major factors, which influence the activity of an enzyme called catalase. Catalase is an enzyme, which is found in many cells, but in highest levels in the liver because the liver often functions to break down toxins present in the blood. Catalase catalyzes the break down of hydrogen peroxide:

 

2H2O2 ------> 2H20 + O2

 

Peroxides can form in the body during respiration, and are chemically reactive, which means that they can chemically modify (and thus render useless) other biological molecules.

Follow this link to get more helpful information about catalase.

Materials and Methods:

At your lab table, you will find everything you need to conduct today's experiments. Each lab station is set up to perform a different section of the Lab exercises (A-F). Check with your lab instructor for updated information about which sections your group is responsible for.

The activities are as follows:

The protocol below describes how to measure the activity of liver catalase by measuring the amount of O2 that is produced when liver catalase is combined with its substrate, hydrogen peroxide (H2O2). You will collect data in terms of the production of one of the products (volume of O2 produced in mL), and you will then convert to enzyme activity units by dividing the volume of oxygen produced by the amount of time you allow the experiment to run. In this lab for ease of calculation, you will measure for one minute. This will give you enzyme activity measured as a rate (mL/min).

For example, after setting up the apparatus as described below, you will then measure the amount of O2 collected in the graduated cylinder after one minute. If the amount of O2 produced after one minute of reaction time is 32 mL, then the enzyme activity is: 32 mL/1 min= 32 mL/min.

For each parts A-F, you will be graphing enzyme activity on the Y-axis (the dependent variable) and the independent variable (parts A-F) on the X-axis. Your instructor will give you clear directions on how to prepare your complete lab report.

Protocol

Work as a team of 4 at your table to perform the experiment. Each group will be assigned one or more specific experiments in lab. You will record your results on the instructor's computer and these results will be made available to you as a link off of the lab webpage or as an e-mail attachment to do your write-up. Each group has to analyze all 6 parts (A-F) of the experiments from the lab's pooled data even though your group only does one or two parts.

 

At your table you will find a small rectangular bottle fitted with a rubber stopper and metal tube (reaction vessel), a 100 ml graduated cylinder and holder, a plastic pan, and a supply of small pieces of filter paper. Fill the pan 2/3 full of tap water, which will quickly become room temperature. Submerge the graduated cylinder to fill it with water. Turn the graduated cylinder upside down, keeping the open end under water, and suspend it upside down in the clamp. Adjust the height of the clamp so the open end of the cylinder is about 2 cm below the surface of the water.

Place a thermometer in the pan and sometime during Part A, record the temperature of the water. Figure 1 shows a picture of the setup.

 

Figure 1: Experimental set up for enzyme lab

For all parts A through F, each reaction vessels will have 3 soaked disks and 10 ml substrate solutions as shown in Figure 2 below:

Figure 2: Reaction vessel

The following procedures are used to obtain oxygen production and are to be repeated for all Sections A-F.

1.   Remove the stopper and lay the reaction vessel on its side on the table.  Then, using forceps, dip three filter disks (one at a time) in the enzyme solution. Remove the excess liquid from the disks by dabbing a corner of the filter paper on a Kimwipe or a paper towel. Next transfer the dampened disks to one interior wall of the reaction vessel. Position the disks in the front bottom half of the reaction vessel (the half nearest the opening).One person in each group should soak and handle all disks for all experiments. Such procedures should be employed in group experiments to assure that key operations are performed exactly the same way.

2.Rotate the reaction vessel so that the disks are on the top side (see picture above) and then add 10 ml of substrate (H2O2) solution.Be careful not to splash H2O2 on the disks. Insert the stopper (with metal tube) in the reaction vessel.

3.Keep the side with the disks upward and carefully place the reaction vessel on its side in the pan of water. The metal tube is placed right beneath the opening of the upside down graduated cylinder.

4.Get the timer ready! Rotate the reaction 180 degrees so the disks will be covered by the H2O2 solution.This is time zero and start the timer now.

5.Measure the oxygen level in the graduated cylinder at 1 minute (from the time the reaction vessel is turned on its side).

6.Remove the disks, then thoroughly rinse and dry your reaction vessel

 

A.INFLUENCE OF CATALASE CONCENTRATION ON TOTAL ACTIVITY

 

Repeat steps 1-6 using various enzyme solution:

1)Three catalase-soaked disks

2)Two catalase-soaked disks and 1 disk soaked in water

3)One catalase-soaked disk and 2 disks soaked in water

4) Three disks soaked in distilled water. The substrate for all four cases is 3 % H2O2 solution.

Graph your results as enzyme concentration (x-axis) vs. Velocity (Vol of O2/min) and explain the relationship, which appears to exist, between the concentration of the enzyme catalase and the breakdown of the peroxide.

 

B. INFLUENCE OF SUBSTRATE CONCENTRATION ON CATALASE ACTIVITY

Repeat steps 1-6 the using three catalase-soaked disks), using different concentrations of substrate, H2O2, as shown below.

1.0%H2O2(Distilled Water)

2.0.3% H2O2

3. 1.5% H2O2

4. 3.0% H2O2.

 

Graph your results as substrate concentration  (x-axis) vs. Velocity (Vol of O2/min) and explain the relationship, which appears to exist between the concentration of substrate and the rate of enzyme activity.

C. INFLUENCE OF pH ON CATALASE ACTIVITY

Repeat steps 1-6 with three catalase-soaked disks, using catalase solutions at the various pH values shown below. Use 3% H2O2 as the substrate for each trial.

5)pH 2

6)pH 4

7)pH 7

8)pH 10

 

Graph your results as pH (x-axis) vs. Velocity (Vol of O2/min) and explain the relationship which appears to exist between pH and catalase activity.

D. INFLUENCE OF IONIC CONCENTRATION ON CATALASE ACTIVITY

Repeat steps 1-6 (using three catalase-soaked disks) using different concentrations of NaCl in the substrate solution.

1.1.5% H2O2 solution containing 10% NaCl.

2.1.5% H2O2 solution containing 2% NaCl.

3.1.5% H2O2 solution containing 0% NaCl.

 

Graph your results as ionic concentration  (x-axis) vs. Velocity (Vol of O2/min) and explain the relationship which appears to exist between the concentration of sodium chloride ions and catalase activity.

 

E. INFLUENCE OF TEMPERATURE ON CATALASE ACTIVITY

Repeat steps 1-6  (using three catalase-soaked disks and 10 mls of 3% H2O2) in each of the different temperature water baths.

1.Three disks soaked in the "boiled" catalase extract (100C) in a room temperature water-bath and reaction vessel with 3% H2O2. Allow equipment to stabilize at room temperature for 2-3 minutes before making run.   This simulates a boiling water bath, without the danger.

2.A water bath that has been chilled with ice to 5C. (Keep adding ice to keep temperature close to 5C. Allow reaction vessel to stabilize in water bath for 2-3 minutes before making the run. Record the temperature.

3.A water bath that has been warmed to 33C. Allow temperature of the reaction vessel to stabilize in the water bath for 2-3 minutes at 33C before making the run. This temperature is about 91.4F, which in the body, is equivalent to a state of hypothermia.

4.A water bath that has been warmed to 37C. Allow temperature of reaction vessel to stabilize in the water bath for 2-3 minutes before making the run. This temperature is about 98.6F, which is equivalent to standard body temperature.

5.A water bath that has been warmed to 41.1C. Allow temperature of reaction vessel to stabilize for 2-3 minutes in the water bath before making the run. This temperature is about 106F, which in the body, is equivalent to a state of hyperthermia.

6. Use the results of the trial from part A, step1 (the catalase reaction with 3 disks at room temperature) for the Room temperature data point requested on the class spreadsheet.

Graph your results as temperature (x-axis) vs. Velocity (Vol of O2/min) and explain the relationship which appears to exist between temperature and catalase activity.

F. INFLUENCE OF ENZYME INHIBITOR ON CATALASE ACTIVITY

Repeat steps 1-6 using 3 disks soaked with catalase at different concentration of copper sulfate.  You will be given five tubes containing the catalase solution, five beakers containing copper sulfate (CuSO4) solution at various concentrations and five weigh boats.

 

Plan your experiment CAREFULLY!!!

Graph your results as copper sulfate concentration (M) (x-axis) vs. Velocity (Vol of O2/min) and explain the relationship, which appears to exist between copper sulfate concentration and catalase activity.

The 0 M copper sulfate data point is the value generated in Part A #1: three disks soaked in catalase.

 

G. INSTRUCTIONS FOR ENZYME PAPER - rough draft is due the week of February 21.

As a group, complete a full scientific paper. This paper will include parts A-F, even though your group only completed part of the experiment.

Here are a couple of internet resources that will help you to write a scientific paper on the experiment.

Writing a Lab Report: a link to an Introductory Biology course at UNCG dealing with writing a lab report.

Writing Lab reports and scientific papers: by Warren D. Dolphin at Iowa State University

Use the Long Island University library website for the proper MLA format for citing your references in your report.

Dr. Garrison's sample lab report

Note 2: Use your text and the internet resources at the beginning of this lab to discuss the basic principles of enzyme function. Also include in your background information about catalase activity. Also, in discussing your results, in the discussion section you will also need to do some research to explain the effect of the various conditions have on catalase activity.

REFERENCES 

Abramoff, P. & Thomson, R. G. 1976. An Experimental Approach to Biology, pp. 65-73. W. H. Freeman and Co.

Starr, C. & Taggart, R. 1981. Biology: The Unity and Diversity of Life, pp. 84-94. Wadsworth Publishing Company.

Weisz, P. D., & Tarp, F. H. 1967. Laboratory Manual for Elements of Biology, pp. 25-32. McGraw-Hill Book Co.

Back to 270 lab manual link