Enzyme Function

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Download a PDF version of this page and print it out prior to attending lab.

Pre-lab Questions

Answer the following questions in your lab notebook before attending lab.

  1. What is meant by rate of reaction?
  2. What effect does raising the temperature of a chemical reaction have on the rate of that reaction? Please draw a graph of this expected relationship in your lab notebook and make certain to label the axes of your graph. Please note that this is a sketch.
  3. How does the concentration of reactants in a chemical reaction change over time? Please draw a graph of this expected relationship in your lab notebook and make certain to label the axes of your graph. Please note that this is a sketch.
  4. How does the concentration of products in a chemical reaction change over time? Please draw a graph of this expected relationship in your lab notebook and make certain to label the axes of your graph. Please note that this is a sketch.
  5. How does the concentration of enzyme in a catalyzed chemical reaction effect the rate of that reaction? Please draw a graph of this expected relationship in your lab notebook and make certain to label the axes of your graph. Please note that this is a sketch.

Overview

Enzymes are proteins which catalyze biological reactions. Some of them are "simple proteins", others are "conjugated proteins"; the latter can be decomposed into the protein group and another complex organic component (the prosthetic group). In either case, the enzyme-catalyzed reaction depends on a transient combination between the enzyme and the substrate or compound whose reactivity is accelerated by the enzyme. This transient combination is called the Enzyme Substrate Complex.

It follows that the velocity of an enzyme-catalyzed reaction will depend on the number of combinations per unit time between the enzyme and the substrate; factors which affect the reactivity of the active sites of combination will also influence the velocity of the reaction. If the amount of substrate is in excess, there should be a straight line relation between the rate of reaction and the amount of enzyme: further, the rate of reaction should rise to a plateau if the amount of substrate is gradually increased in the presence of a constant amount of enzyme. Like all chemical reactions there should also be dependence on temperature. The temperature pH may be particularly important in enzyme-catalyzed reactions because of their effects on the stability of the enzyme and the fact that enzymes, like other proteins, are multivalent dipolar ions which dissociate in accordance with the pH of the medium.

This exercise investigates the naturally occurring enzyme peroxidase. Peroxidase is a heme-containing enzyme found in peroxisomes and can be obtained from a variety of plant tissues. Each cell uses oxygen in its metabolism. A resulting by-product, H2O2, is highly toxic and must be removed immediately by peroxidase or other enzymes before the cell is damaged. The reaction that will be monitored is:

peroxidase
RH2 + H2O2 --------------> R + 2H2O

where RH2 stands for a variety of H donors and R for the form of the molecule after having donated hydrogen.

It is impossible to see the reaction in vitro because all of the reactants are colorless. We use the reducing agent, guaiacol, as the H donor, because it changes color when it loses hydrogens, forming a brown product, tetraguaiacol. Because the process now involves a color change, it is possible to use a spectrophotometer to monitor the rate of change. The absorbance is a direct measure of the amount of substrate hydrolyzed.

This lab includes the formulation of hypotheses to be tested by the experiments. In forming an hypothesis, the assumptions are stated and a tentative explanation proposed that links possible cause and effect. A key aspect of a hypothesis, and indeed of the modern scientific method, is that the hypothesis must be falsifiable, i.e., if a critical experiment were performed and yielded certain information, the hypothesis would be declared false and discarded because it was not useful in predicting any natural phenomenon. Science advances as a result of the rejection of false ideas expressed as hypotheses and tested through experiments. Hypotheses that over the years are not falsified and which are useful in predicting natural phenomena are called theories or principles-for example, the principles of Mendelian genetics.

Hypotheses are made in mutually exclusive couplets called the null hypothesis (Ho) and the alternative hypothesis (Ha). The null hypothesis is stated as a negative and the alternative as a positive. For example, when crossing fruit flies a null hypothesis might be that the principles of Mendelian genetics do not predict the outcomes of the experiment. The alternative hypothesis would be that Mendelian principles do predict the outcome of the experiment. As you can see, rival hypotheses constitute alternative, mutually exclusive statements; both cannot be true. The purpose in proposing a null hypothesis is to make a statement that could be proven false if data were available. Construct a null hypothesis and an alternative hypothesis for each experiment before coming to lab.

Procedure

Preparing an Extract Containing Peroxidase

  1. Weigh 8 g of peeled turnip tissue on a balance.
  2. Homogenize the tissue by adding it to 300 ml of cold (4°C) 0.1 M phosphate buffer at pH 7. Blend it for 20 seconds at high speed in a cold blender. Filter the extract through several layers of cheesecloth. Keep the extract on ice. The extract will keep for about ten hours in a refrigerator.

Standardizing the Amount of Enzyme

Directions for using the spectrophotometer are available here.

Directions for creating dilutions are available here.

The extract you prepared contains hundreds of different types of enzymes, including peroxidase. The activity of each enzyme will vary, depending on the size and age of the turnip; the extent of the tissue homogenization; and the age of the extract. Only peroxidase, however, will react with H2O2.

To demonstrate that the amount of enzyme influences the rate of the reaction and to determine the correct amount of extract to use in future experiments, a trial run should be performed in which the amount of enzyme added is the only variable.

State null (Ho) and alternative (Ha) hypotheses that relate the rate of reaction to the amount of enzyme added. To test your Ho, use the following directions to set up the chemical reactions and to conduct experiment:

  1. Label two test tubes as follows: buffer, pH 5; buffer, pH 7. Fill each about half full with the appropriate stock solution. Label two pipettes to correspond with the solutions. Keep the turnip extract in a beaker on ice. The 25 mM guaiacol and 10 mM hydrogen peroxide solutions are in brown bottles.
  2. Number three test tubes from 1 to 3. The contents of the tubes will be:
    1. Control with no extract to be used in calibrating the spectrophotometer
    2. Substrate and indicator dye
    3. Extract solution

    Tubes 2 and 3 will be quickly mixed together when it is time to measure a reaction. Mix them only when you are ready to measure that reaction in the spectrophotometer. The exact quantities to be added to each tube are listed in Table 1.

    Table 1. Mixing table for trial run to determine extract concentration (all values in ml).

    TubeBuffer (pH 5)Buffer (pH 7)H2O2ExtractGuaiacolTotal Volume
    1 Control4.01.02.001.08
    21.002.001.04
    32.01.001.004
  3. Add stock solutions to each tube using the appropriate pipettes. Use of the wrong pipette will cross contaminate your reagents and introduce errors into your subsequent experiments in this exercise.
  4. Adjust the spectrophotometer to zero absorbance at 500 nm. Pour the contents of test tube 1 into a cuvette. This tube is used to "blank" the spectrophotometer, so that any color caused by contaminants in the reagents will not influence subsequent measurements.
  5. Within your group, one person can be a timer, another, a spectrophotometer reader, and another, a data recorder. Wipe a cuvette with lens paper and handle it by only the top 1/4th.
  6. Pour the contents of text tube 2 into test tube 3. Quickly pour the mixture into the clean cuvette and immediately place the cuvette in the spectrophotometer. Immediately start the timer and read the absorbance. This is your 0 second reading.
  7. Read the absorbance at 20-second intervals from the start of mixing. If you are a little late in reading the meter, record the absorbance and change the table to show the actual time. Record your measurements in a table. After 120 seconds remove the tube from the spectrophotometer and visually note the color change. Discard the solution.
  8. Using 1 ml of turnip extract should give a linear absorbance change from 0 to around 1 in approximately 120 seconds. If this is so, then this will be the standardized amount of extract. If this amount of extract does not produce the desired change in absorbance, then repeat the experiment with different amounts of the extract and the pH 7 buffer until the appropriate change in absorbance is attained.
  9. Repeat the experiment with the appropriate amount of extract but record the absorbance at 0, 15, 30, 45 and 60 seconds. The change in absorbance from 15 and 45 seconds is doubled to give enzyme activity in ΔA500/min.

What is the effect of changing enzyme amount on the reaction?

  1. Repeat the previous procedure using solutions with ½ the amount of turnip extract and 2X the amount of extract determined in the standardization. Adjust the amount of buffer in the tubes to keep the total volume at 8 ml. For example, if the standardized amount of extract is 1 ml then use ½ and 2 ml in this part. See Table 2 as an example.

    Table 2. Mixing table for effect of changing enzyme amount. (all values in ml).

    TubeBuffer (pH 5)Buffer (pH 7)H2O2ExtractGuaiacolTotal Volume
    1 Control4.01.02.001.08
    41.002.001.04
    52.51.000.504
    61.002.001.04
    71.01.002.004
  2. Mix the contents of tubes 4 and 5, pour into a cuvette, and repeat your measurements for 60 sec at 15-second intervals. Record the results and calculate the ΔA500/min.
  3. Now plot the time and absorbance values for each extract volume. The abscissa (X-axis) should be the independent variable and the ordinate (Y-axis) the dependent variable. In this experiment, which is the dependent variable?
  4. Plot all three tests on the same coordinates using different plotting symbols. Plot all graphs with Excel or another graphing program. Use a regression line (trend line) to show the line of best fit.
  5. Do you accept or reject your Ho (null hypothesis) regarding rate of reaction and amount of enzyme? Why?

What is the effect of pH?

State null (Ho) and alternative (Ha) hypotheses that relate change in enzyme activity to the pH of the solutions used.

To determine the effect of pH on peroxidase, perform the following experiment. Your instructor will supply buffers at pHs of 3, 5, 7, and 9. Number nine test tubes 1 through 9. Set up pH effect tests by adding the reagents described in Table 3.

Table 3. Mixing table for pH experiment (all values in ml).

pHTubeBufferH2O2ExtractGuaiacolTotal Volume
51 Control6.0 (pH 5)01.01.08
32
3
0
4.0 (pH 3)
2.0
0
0
1.0
1.0
0
3
5
54
5
0
4.0 (pH 5)
2.0
0
0
1.0
1.0
0
3
5
76
7
0
4.0 (pH 7)
2.0
0
0
1.0
1.0
0
3
5
98
9
0
4.0 (pH 9)
2.0
0
0
1.0
1.0
0
3
5

After adjusting the spectrophotometer with the contents of test tube 1, mix pairs of tubes one at time (2 and 3, 4 and 5, 6 and 7, 8 and 9) and measure absorbance changes at 15-second intervals for 60 seconds for each mixed pair. Record and graph the results. The slopes of the linear portions of these curves are a measure of enzyme activity. Does activity vary with pH? What are the units of activity? What is the optimum pH?

These results should be graphed. The slopes of the linear portions of these curves are a measure of enzyme activity. Does activity vary with pH? What is the optimum pH?

To show clearly this relationship, you should prepare another graph after lab. Determine the absorbance change per minute (slope) at each pH from your linear graphs.

Do you accept or reject the Ho regarding pH effects on enzymes? Why?

What is the effect of boiling on peroxidase activity?

Most proteins are denatured when they are heated to temperatures above 70°C. Denaturation is a nonreversible change in a protein's three-dimensional structure. If the shape of an enzyme is significantly altered, what do you predict will happen to measured enzyme activity?

State null (Ho) and alternative (Ha) hypotheses that relate heat treatment of an enzyme (boiling) to the expected effect on that enzyme's activity.

To perform the experiment that tests your null hypothesis, follow these directions:

Add 3 ml of extract to a test tube and place it in a boiling water bath. After five minutes, remove the tube and let it cool to room temperature. Number three test tubes and add reagents as called for in mixing Table 4.

Table 4. Mixing table for effect of changing enzyme amount (all values in ml).

TubeBuffer (pH 5)Buffer (pH 7)H2O2ExtractGuaiacolTotal Volume
1 Control5.01.0011.08
201.02.001.04
32.01.001.004

Use the contents of tube 1 to blank the spectrophotometer. Mix the contents of tubes 2 and 3, pour the mixture into a cuvette, and read the absorbance at 15-second intervals for 60 seconds. Record the results in a table. Compare the activity of peroxidase after boiling to the activity of peroxidase kept at room temperature and pH 5. How did boiling affect the activity?

Consult your text to see what kinds of chemical bonds are disrupted in proteins heated to 100°C. Describe these bonds.

Do you accept or reject the Ho made at the beginning of this experiment? Why?

How does temperature affect enzyme activity?

To determine the effects of temperature on peroxidase activity, you will repeat the enzyme assay in water baths at four temperatures:

  1. In an ice bath at approximately 4°C
  2. At room temperature (about 23°C)
  3. At 32°C
  4. At 48°C

State null (Ho) and alternative (Ha) hypotheses that relate change in enzyme activity to the temperature of the solutions used.

To test your Ho, you should use the following directions to set up the reactions and conduct the experiment. Number nine test tubes in sequence 1 through 9. Refer to table 5 for the volumes of reagents to be added to each tube.

Table 5. Mixing table for temperature experiment (all values in ml).

TemperatureTubeBuffer (pH 5)Buffer (pH 7)H2O2ExtractGuaiacolTotal Volume

1 Control6.0001.01.08
4°2
3
1.0
2.0
0
1.0
2.0
0
0
1.0
1.0
0
4
4
23°4
5
1.0
2.0
0
1.0
2.0
0
0
1.0
1.0
0
4
4
32°6
7
1.0
2.0
0
1.0
2.0
0
0
1.0
1.0
0
4
4
48°4
5
1.0
2.0
0
1.0
2.0
0
0
1.0
1.0
0
4
4

Preincubate all the solutions at the appropriate temperatures for at least 15 minutes before mixing. After reaching temperature equilibrium and adjusting the spectrophotometer with the contents of test tube 1, mix pairs of tubes (2 and 3, 4 and 5, 6 and 7, and 8 and 9) one pair at a time and measure changes in absorbance for 60 seconds at 15-second intervals for each temperature. The temperatures will not remain exact, but the effects can be overlooked.

Note: after the spectrophotometer is adjusted, the room-temperature experiment can be performed immediately while the other tubes temperature-equilibrate.

Record changes in absorbance for each temperature in a table.

These results should be graphed. The slopes of the linear portions of these curves are a measure of enzyme activity. Does activity vary with temperature? What is the optimum temperature?

To show clearly this relationship, you should prepare another graph after lab. Determine the absorbance change per minute (slope) at each temperature treatment from your linear graphs.

Plot the activity (slope) values as functions of temperature. Do you accept or reject the Ho stated earlier about the effect of temperature? Why?

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