Objectives
to test for the presence of enzymes in a variety of materials
to show the impact of various chemical and physical factors on enzyme activity
Introduction
The cell is a vast biochemical factory carrying out metabolic activities. Catabolic reactions provide raw materials and starting energy for various anabolic activities. The rate of these reactions is controlled by biological catalysts, the enzymes.
Catalysts are not used up in the reaction, and do not furnish energy for the reaction. Catalysts merely affect the rate of the reaction by reducing the amount of activation energy required. This speeds up the rate at which reactions take place. All of these cellular biochemical reactions are thermodynamically possible. The use of the enzymes merely speeds up these reactions.
Enzymes are proteins which may require the presence of a coenzyme such as a vitamin or a cofactor such as an inorganic ion (Mg++, Mn+, Cl-) in order to operate properly. Changes in the hydrogen ion concentration (pH), and in temperature, may change the configuration or shape of the enzyme molecule. If this results in altering the "fit" of the enzyme to its substrate, the speed at which the reaction occurs may be slowed or the reaction may not occur at all. Extremes of pH, and high temperatures, DENATURE enzymes which stops their action.
Enzymes combine with the reactant molecules (SUBSTRATE) and bind them closely to one another. The three-dimensional shape of the enzyme molecule must be complemantary to the shape of the substrate. This is known as ENZYME SPECIFICITY. The reaction can proceed rapidly without much activation energy. Most of these reactions are essentially reversible, and the direction in which the reaction goes depends on the concentration of the reactants in relation to the concentration of the products. Standard reactions are shown below:
enzyme + substrate <=> enzyme-substrate complex <=> enzyme + product
Note that the enzyme is freed and available for reuse.
The rate at which the reactants are converted to products is controlled by pH, enzyme concentration, temperature, substrate concentration, and product concentration.
PURPOSE
The purposes of this investigation are to study multiple aspects of enzyme activity including the effect of pH on the rate of reaction of the enzyme catalase. Molecules of hydrogen peroxide (H2O2) serve as the substrate for the enzyme. In this investigation, a yeast suspension is used to provide the enzyme catalase. Other materials such as crushed potatoes, onions, or carrots could be used. Small filter paper circles are dipped into a catalase suspension and dropped into a very dilute hydrogen peroxide solution. The filter paper sinks to the bottom due to its density. Eventually the paper will float to the surface. The time required for this to occur depends directly on the rate of the reaction occurring between enzyme and substrate.
Follow this link to see the complete investigation done by Mark Radcliffe of the effect of substrate concentration on the rate of reaction of catalase.Additional sources of enzymes will also be looked at.
MATERIALS
Hydrogen peroxide solution (available at your local grocery or pharmacy)
packets of yeast and other food goodies
Scissors or razor blades or a sharp knife
millimeter ruler
Stopwatch or a watch with a second hand
Forceps or tweezers
Filter paper(#3) or coffee filters
Paper labels or masking tape
pens
three hole paper punch
containers to hold materials
PROCEDURE
- Mix the yeast in a beaker of warm water. Allow about 30 minutes for the yeast to activate. Label this mixture "Catalase." You could also mash up some potatoes or onions to create a catalase solution. Take an equal amount in grams of potato or onion, e.g. 50 grams, and add an equal amount of water in milliliter, e.g. 50 ml and mash them up or use a blender. Filter the resulting mash through a coffee filter. You can then dilute this filtrate as needed.
- Cut #3 filter paper into circles or squares that are 5 mm on a
side. You can also use a three hole or single hole paper punch to create equal size circles from coffee filters (#4) (decent grade of paper). Avoid excessive contact with the paper and be sure that
hands are clean or wear gloves. Oils from the hands can alter
the paper's ability to absorb the catalase solution.
- Prepare various dilutions, such as 25% or 50%, of hydrogen peroxide in water (tap is OK) in the 500 mL graduated cylinder. (No graduated cyclinder? A cup is 240 ml and a teaspoon is 5 ml. The rest of the math is up to you.) To test the strength of the solution, pour about 50 mL of the hydrogen peroxide into a beaker. Using forceps, dip a filter paper circle into the catalase mixture. Allow the paper circle to absorb the material for about 5 seconds. Drain the excess by holding the paper disc on the side of the tube or by touching a clean paper towel. Drop the filter paper circle into the beaker. The paper should sink completely to the bottom and then float to the top in about 40 seconds. If it takes less time, add water to further dilute the peroxide. If it takes too much time, add more hydrogen peroxide and test again. You will probably have to add water.
- Record the time it takes for the filter paper to float to the
surface (i.e. horizontal). You should begin the timing when
you drop the paper into the beaker. Create a graph that shows the rate differences as they vary with concentrations of the substrate. Submit this graph with the answers to the questions.
- You could test a food for catalase activity by merely taking a small cube of the material, such as potatoes, onions, or carrots and then some other materials such as a piece of chicken or turkey, and setting them in a tube to which full strength hydrogen peroxide is added
- A quick way to determine the effect of pH on catalase activity is to cover a potato cube with vinegar and another with ammonia and carefully add hydrogen peroxide to each tube. Create a control tube by adding water to a potato cube and then adding the hydrogen peroxide. BE CAREFUL WORKING WITH THE AMMONIA. DO NOT LOOK DIRECTLY INTO THE CONTAINER WHEN YOU ARE ADDING THE HYDROGEN PEROXIDE. LOOK AT THE TUBE OR CONTAINER FROM THE SIDE.
Assignment:
1. Explain, using complete, grammatically correct sentences
why the filter paper rose to the surface. Include the formula showing the reaction between catalase and its substrate.
2. Devise an experiment, using the same or any other enzyme and substrate that will measure the effects of temperature on rate of reaction and can be done at home. This experiment should make it possible to determine the optimum temperature for catalase activity. You may need to try this out before you report your materials, preparation and procedures.
3. Surf the Internet, using the Google Search Engine, and find two or three other enzyme labs that use various types of fruits as the source of proteolytic enzymes. Provide the URL's for these labs.
Additional Sources
Clariana, R.B. (1991) pH and rate of enzymatic reactions.
The American Biology Teacher, 53(6), 349-350
Lennox, J.E. & Kuchera, M.J. (1986) pH and microbial growth.
The American Biology Teacher, 48(4), 239-241.