ENZYMATIC REACTIONS
Ruth Logan, 2003
The main purpose of these exercises is to illustrate the properties of enzymes, those
protein molecules that control and direct metabolic activities. We will measure the activity
of a model enzyme under varying conditions, including some that will inhibit activity, and
discuss why each example behaves as it does. In a self-design experimental portion of
the lab, we will test the effects varying conditions, such as the concentrations of enzyme
or substrate, have on the rate at which reactions proceed. The data produced in this part
will be analyzed in a lab report.
Phenolases: A Model Enzyme System
There is a class of related enzymes known as phenolases. These are copper-containing
enzymes that catalyze the oxidation of phenols (compounds that have hydroxyl groups
attached to a benzene ring), to the corresponding quinone (compounds that are cyclic
diketones). Some of these enzymes, such as tyrosinase, monophenol oxidase, diphenol
oxidase, or catecholase, are named for the specific substrate they oxidize. In this
experiment you will measure phenolase activity by observing the production of pink/gold
color when catechol (the substrate) is placed into solution with a phenolase (catecholase)
from potatoes.
Catechol is oxidized by the enzyme to form 0-benzoquinone (a quinone) that is then
converted through a series of spontaneous reactions to produce a heterogeneous group
of polymers called melanins. As the polymers gets larger, their colors deepen from pink/
gold through orange-brown and finally to an intense brown-black color. The larger
molecules are less soluble in water so eventually precipitate from the solution. The
reaction pathway is shown below.
Phenolase
spontaneous
spontaneous
Catechol -----> 0-benzoquinone---> ---> pink/gold melanin ---> ---> brown melanin
+1/2 O2
+H2O
You will be able to measure the amount of phenolase activity by measuring the
concentration of a product of the reaction. Light energy of wavelength 475 nm is
absorbed by some relatively long-lasting melanin intermediates that are pinkish-gold in
color, so the absorbance measured in a spectrophotometer at this wavelength is directly
proportional to their concentration. Since they are intermediates in a series of reactions, if
the experiment goes on long enough, these compounds will eventually be converted to
the next deeper colored (brown/black) intermediate and will absorb less energy at 475 nm.
That is, they will "disappear" from your measurements.
Melanin is the pigment that colors hair, skin, and eyes in animals. The phenol that leads
to melanin production in animals is tyrosine and therefore the phenolase that acts on it is
usually called tyrosinase. The Himalayan rabbit has white fur except at the tips of its tail,
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Enzymatic Reactions
Logan, 2003
feet, ears, and nose, where the fur is dark. The explanation for this color pattern is that
the tyrosinase of the rabbit is temperature sensitive. Therefore, over most of its body the
temperature is high enough to inactivate the enzyme. Only at its extremities is the
temperature sufficiently low for the tyrosinase to be active with the resulting production of
the darkly pigmented melanin. Humans and other animals that lack tyrosinase are called
albinos and lack of pigment in skin, hair, and iris of the eye.
When potatoes, apples, bananas, sweet potatoes, or mushrooms are injured or peeled,
they turn brown because their phenolases are exposed to oxygen and can oxidize
substrates already present in their cells. It is the phenolase from potatoes that we are
using in this experiment.
Fresh potato extract containing the enzyme phenolase will be provided for each class
session by using the following procedure: Peeled diced potatoes were homogenized
(ground up) in a Warring blender with 200 ml of COLD phosphate buffer pH 6.0 for 1 to 2
minutes. The resulting mixture was filtered through a large COLD funnel lined with
several layers of cheesecloth into a COLD beaker. The cheesecloth and solid waste was
discarded. The supernatant potato extract was poured into centrifuge bottles that were
balanced on the pan balance and centrifuged for 5 min. at 4000 rpm in the centrifuge.
The supernatant from these tubes was poured into a COLD Erlenmeyer flask and kept on
ice for use in the lab.
This potato extract contains polyphenol-oxidase, the enzyme to be studied today PLUS
every other soluble enzyme from the cytoplasms of the potato cells.
The structures of the three phenolic compounds used in this experiment are shown below:
OH
OH
OH
OH
OH
Catechol
Hydroquinone
Phenol
These phenolic compounds may be irritating to the human skin, and
may damage internal organs if ingested. While the compounds are
used here in a very dilute form, use care in handling them: Don’t get
them on your skin. Don’t take them into your lungs or mouth. Dispose
of them in the container as directed in the laboratory.
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Logan, 2003
Assay for enzyme activity:
1.
2.
3.
4.
5.
Prepare a 5 ml reaction mixture in a test tube as indicated below.
Start to keep time when substrate and enzyme are first both present in the solution.
Mix rapidly by pouring the solution into a cuvette.
Measure the absorbance at 475 nm in the spectrophotometer 30 seconds after t=0.
Take subsequent absorbance readings as indicated.
Since a melanin product absorbs light of this energy, the difference in the absorbance of a
solution over any time period is directly proportional to the amount of melanins produced
in the solution during that time period. Since melanins are a product of the reaction
catalyzed by the enzyme phenolase, the rate at which melanins are produced is an
indication of the rate of this reaction.
Throughout these experiments, look at the solutions and record their appearance. Are
they turning reddish brown in correspondence to the absorbance reading? If not, look for
other factors that might affect the apparent absorbance reading. Precipitates (including
cloudiness), air bubbles, and fingerprints on the cuvette are all quite famous for impeding
or scattering the light. These observations will be very valuable when you attempt error
analysis.
Experiment
The format for this experiment is to first perform some “quick and dirty” trials in order to set
up a plan for an experiment. These initial trials are “quick” because they take just five
minutes each. They are “dirty” because they rely on just two measurements of the
absorbance (30 seconds and 5 minutes), which gives a low precision result. We can get
an idea of what is happening, but no rigorous observations will have been made.
In order to study the real effects of reaction conditions on the activity of the enzyme, we
need to design a series of experiments that are much more precise and frequent in their
measurements. Instructions for that part of the experiment follow.
A. Varying the reaction conditions:
Prepare the samples shown in the table below. The standard reaction mixture is to be
compared with controls and with variations in substrates, pH, and solution environment.
For this first section, add the potato extract LAST and start recording time when it is
added. Read the absorbance of each tube 30 seconds after the reaction has started and
again after it has continued for 5 minutes. If a precipitate forms, spin with a counter
balance in the centrifuge, decant, and then measure absorbance.
Record any
observations (color change, precipitate, etc.) in the space indicated, and state your
conclusions as to what occurred in each sample.
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Logan, 2003
Materials to add to each sample:
0.1%
Catechol
(substrate)
Add mL
Phos Buff
pH 6.0
Pot extract
(enzyme)
Other
Abs30 sec
Add mL
Add mL
Add mL
Abs5 min
Δ Abs 4.5 min
Observations
Standard Reaction Mixture:
1
3
1
Control #1: no enzyme
1
4
Control #2: no substrate
4
1
Alternative substrates: These compounds are similar to catechol in structure.
3
1
1 phenol
3
1
1
hydroquinone
Variations in pH: acid and base
1
1
3 HCl
1
1
3 NaOH
(Look at this one’s color BEFORE and after you add the enzyme!)
Solution environment: Mercury is a well-known poison. Ascorbic acid is easily oxidized
and uses up oxygen in the environment.
1
1
3 HgCl2
1
1
3 ascorbic
acid
B. Modification of the Enzyme:
For these samples, add the SUBSTRATE last, just as you start the time.
Ethanol, low polarity solvent: add to enzyme and wait 10 minutes to add substrate.
1
1
3 ethanol
Protease (an enzyme that hydrolyzes peptide bonds): add and wait 10 minutes as above.
1
1
3 protease
Phenyl Thiourea – a well-know chelating agent. Add a pinch to bind up the copper. Wait
10 minutes as above.
pinch
1
3
1
Boiling water: boil a sample of enzyme and buffer for 10 minutes before adding substrate.
1
3
1
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Logan, 2003
C. Time Course of an Enzymatic Reaction
This is the method you will use to measure time course of reaction in the self-design
experiment that follows. You will take the rate of reaction from the slope of an early part
of the curve, and will use the level later part of the curve to help analyze error.
Prepare a test tube containing the same components as the standard reaction mixture.
Mix by pouring into a cuvette. Read the absorbance of this sample every 30 seconds
from 30 seconds after you add the enzyme until at least 20 minutes or until the
absorbance has leveled off. Plot the absorbance vs. time on a graph and sketch the
curve that the points describe. Explain what is occuring in each part of the curve.
D. Self-Design Enzyme Experiment and Lab Report
You will write a full LAB REPORT for the experiment you are to design and conduct using
the outline presented below. Now would be a good time to re-read Writing The
Scientific Paper, Section 2, near the beginning of this lab manual.
Design of the Experiment
1. Choose a variable that might affect enzyme activity and that you can obtain and
use in the laboratory. One of the following variables would be good, or make one
up:
enzyme concentration
substrate concentration
pH
temperature
concentration of a possible inhibitor (competitive or non-competitive)
2. Prepare an hypothesis stating the effect you think your chosen variable will have on
the RATE of this reaction. Be sure your hypothesis is testable: it should describe
the shape of a graph of rate of reaction vs your variable. (This actually appears in
the Introduction of your report, but you have to write it in order to plan your
experiment.)
3. Plan a set of at least 6 experimental samples in which all factors are kept constant
except the one you are investigating. Show their composition in a table if that is
the easiest way to communicate this information.
4.
Plan a timetable for preparing these tubes according to a schedule so that the
absorbance at 475 nm can be read for each tube at 30-second intervals from 0 to
20 minutes (or longer if leveling off has not occurred) after combining the tube's
contents. Be sure you prepare a table to collect your data before you come
to class.
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Logan, 2003
The Lab Report
Title
Describe the experiment done including its concept and the names of all relevant
variables. Remember that titles usually do not contain a verb.
Introduction
This is the first time you have written an Introduction. It should start by describing the
ISSUE of this experiment, that is, the principal question or idea around which all the
experimentation and analysis will be centered. Usually you will describe a MODEL for the
specific relationship you are studying which you are proposing or you describe from
references you cite. Be sure your model develops a reason WHY the relationship you
propose would make sense. In describing your model, imagine you are inside the test
tubes observing the molecules in action. Finally translate this model into a specific
hypothesis that describes the shape of a graph. Introduce the enzyme system being used
in this section only if it is relevant to the issue you are studying.
Materials and methods
This section must include a complete description of your methods and materials.
Introduce the enzyme system being used (if it has not already been done in the
introduction) and explain how you will measure change in the system. Then use the plans
you developed for this experiment to describe your methods. If you have a Table 1, it
should be referred to in the text and placed in the report near this section. (Certain
experimental designs do not require a Table here).
Results
Use the data in your experimental table to graph the results of absorbance vs time for the
six or more samples you ran (this is Figure 1). You can place all the lines on one graph if
you do it neatly and make it possible to see all data points. Label the curves in a
meaningful way (not “sample 1”, etc.) (If your curves overlap too much, you may split the
data into two or more graphs).
Consider the title and caption you write for this graph. The title should focus the concept
of the graph and the caption should describe the source of the data, the use to which the
data will be put, and any general trends or points that will need special discussion.
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Logan, 2003
To find the rate of reaction for each sample, measure the slope of each curve in Figure 1
during an early part of the experiment. In order to compare these curves, you need to use
the same time period for all of them.
Prepare a graph of your independent variable vs. rate of reaction (Figure 2). This graph
should provide the test for your hypothesis and should have one data point for each curve
in Figure 1. Again take care in writing the title and caption for the Figure.
Write a TEXT section that briefly introduces your results. Include a description of how you
derived Figure 2 from Figure 1 and explain why that method (and time period) was
appropriate. Name the curve shapes you have drawn for each figure and show by error
analysis that they can be accepted for discussion. Don't forget that the text precedes the
figures in the final presentation of the report.
Discussion
Discuss the meaning of the shape(s) of the curves in Figure 1. What do these curves
show? You should describe possible events that might be occurring in the test tube for
each portion of the curve and relate each part of your explanation to specific evidence in
the data that led you to propose that idea. Look at all parts of the curve to see if they
support your explanation for each individual part. When there is not enough data to be
sure, speculate about what is occurring and present your reasoning.
Discuss the shape of Figure 2 in light of your hypothesis. Does it support or fail to support
your hypothesis? How does the variability in the data influence your reasoning in testing
your hypothesis? Can you see through the data to the underlying issue to see if your
samples represent the behavior you proposed in your introduction? Analyze or speculate
about the theoretical explanation for the observed data in Fig. 2 whether it supports your
hypothesis or not. This section of the discussion is very important because it connects
your experiment to actual experience in the real world. After all, science is supposed to
study real phenomena!
Conclusion
Restate your hypothesis in a manner that shows your conclusions including the
uncertainty in them. Show ideas for further testing of your speculations. Be sure that this
section agrees with your previous discussion!
Literature Cited
If you cited any literature in your paper (and you should have), you must now report their
references correctly. Don't forget that you MUST cite a reference when you make a
statement of fact not investigated in this paper. Use the APA style described in Section 2
of this lab manual both in this section and in the body of the report.
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