16 Jun Introduction to Cell and Molecular Biology Biology 211 Lab 6 and 7
Question
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Introduction to Cell and Molecular Biology
Biology 211 Lab 6 and 7
Enzymes and Enzymatic Reactions
All living things interact with the environment, use energy, and give off waste.
To carry out these and other life processes, biochemicals must be made,
transformed and eventually broken down.
These biochemical reactions are rapid, specific and occur at temperatures
compatible with life. What makes these reactions possible are a class of globular
proteins called enzymes. Without these enzymes, life processes are not possible.
Learning Objectives:
Upon completion of this lab you should be able to:
1. Define:
a. Enzyme
b. Catalyst
c. Substrate
d. Product
e. Active site
f. Activation energy
g. Blank
h. Control
i. Reaction velocity
j. Reagent
k. Slope
l. Competitive inhibitor
m. Non-competitive
inhibitor
2. Describe the physical characteristics of an enzyme.
3. What environmental factors affect enzyme activity and how do they exert their
effect?
4. Use your pipetting and measuring skills to set up an experiment to test
enzyme activity.
5. Collect absorbance data using a spectrophotometer.
6. Construct a line (“connect the dots”) graph of the scientific data using
Excel™.
7. Interpret a graph of the data and compare to other graphs.
8. Understand why is a blank important in using the Spectrophotometer.
9. Understand why is a control important in experiments determining enzyme
activity.
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Introduction to Enzymes:
Enzymes have several important characteristics:
? Enzymes are able to take a specific biomolecule called substrate (or substrates
because some enzymes use more than one substrate) and change it into
another specific biomolecule called product (or products).
? Enzymes have what is called an active site. An active site is a pocket or cleft in
the protein that is shaped so that it specifically fits a certain substrate. It is here
that the substrate is converted into a product.
? Enzymes are biological catalysts. They participate in a biological reaction and
guide the reaction, but they are not changed by the reaction. Since they are not
changed by the reaction they are used over and over to carry out the same
reaction.
? Enzymes speed up reaction rate by decreasing the activation energy required
to start the reaction. Activation energy is the energy required to break certain
bonds in the substrate so that other bonds can form. The formation of these new
bonds results in the formation of the product.
? Enzymes, like all proteins, are affected by the environment. Changes in
temperature, pH and the ionic strength of the surrounding solution all affect
enzyme activity. For example, there are some bacteria that have adapted to life
on coal and iron ore “mine tailings”. Tailings are the materials left over after the
coal or iron ore has been removed. When mixed with water, these tailings
commonly have a pH of 2.5 or less and yet Thiobacillus bacteria live quite
happily on it.* What do you think the optimal pH for activity of the enzymes of
these bacteria will be? If you said about pH 2.5 you have the idea! So what do
you think would happen if you tried to grow these “acidophilic” bacteria at a
neutral pH? The answer? Certain death, because the enzymes of acidophilic
bacteria are not adapted a pH of 7. However, most organisms have enzymes
that function best between pH 5 and pH 9.
*Run off from these tailings is a serious source of pollution in mining districts of the
Eastern US. You may be interested to know that there are also bacteria that thrive at
excessive temperatures and highly salty conditions by having enzymes that have
evolved in these extreme environments.
? Certain chemicals, as well as environmental factors can also inhibit enzyme
activity. Some chemicals inhibit enzymes by attaching reversibly to sites on the
enzyme other than the active site (allosteric sites) resulting in a conformational
change to the enzyme so that the enzyme is unable to function. These inhibitors
are known as non-competitive inhibitors because they are able to inhibit an
enzyme without competing with the normal substrate for the active site.
? Other chemicals are so similar to the enzyme substrate that they can bind to the
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active site of the enzyme. Since the chemical is not the normal substrate, it can
not be converted into the product. These particular chemicals do not
permanently bind to the enzyme and therefore they inhibit the enzyme by
competing with the normal substrate for the active site. You can probably guess
that these inhibitors are called competitive inhibitors.
So what enzyme are you going to use in today’s experiment? Since all living
things use enzymes, there are a wide variety of enzymes in nature. For example, there
are 100,000 or more available from the human body alone. But the enzyme we will use
is called alkaline phosphatase. It is found in many different organisms and it catalyses
the removal of a phosphate group from DNA molecules. The reaction works something
like this:
Alkaline Phosphatase
DNA with phosphate attached DNA without phosphate attached + phosphate
(substrate) (products)
You will not use real DNA as a substrate in this experiment because it is difficult to
measure the removal of the phosphate group. We will use another compound called
paranitrophenyl phosphate as the substrate. The reaction looks like this:
Alkaline Phosphatase
Paranitrophenyl phosphate Nitrophenyl + phosphate
(colorless substrate) (yellow product)
The reason we use this reaction is that the removal of the phosphate group from
the substrate (paranitrophenyl phosphate) can be easily measured because the
paranitrophenyl phosphate is colorless and one of the products, nitrophenol, is a bright
yellow color. For this reason it is easy to follow the progress of this reaction with the
spectrophotometer. The Spec 20 is used to measure the change in absorbance as the
reaction proceeds. In this case, as more nitrophenol is produced the solution becomes
a darker yellow and the absorbance increases.
One note about nitrophenol. It is a possibly hazardous material and contact with
skin should be avoided. Wash thoroughly if contact does occur.
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The Experiments:
Enough background, let’s talk about the experiment we are going to do today!
Part I: You are going to set up reactions in test cuvettes which can measure the
formation of product by the enzyme. You will use 5 different concentrations of the
enzyme to determine the optimal concentration of the enzyme to use in further
experiments. You will correctly set up the reagents, run the experiment and graph the
results. Then you will compare the results of the 5 different samples and choose the
optimal enzyme concentration.
Part II: In Part I you found out how much enzyme to use and have seen how well it
works under “normal” conditions. Next you are going to change some of the
environmental parameters and see how that affects enzyme activity.
Experimental Procedures
(all experiments are run at room temperature except where noted)
Part I: Find the optimal concentration of enzyme
Read all of Part I. You should do this before starting any experiment. It allows
you to anticipate what is coming up and lets you avoid nasty surprises.
Blanks and controls are quite important in experiments. The reason for having
blanks and controls are several. Firstly, when using the spectrophotometer you are
trying to measure the formation of product in the experimental cuvettes. Unfortunately
there are several other reagents in the cuvettes that are necessary for the reaction to
occur (like the buffer, the substrate, and the enzyme itself) that are not the chemical
that we are trying to measure. We are only interested in changes in the absorbance
due to the substrate being changed into product. Therefore, if we can exclude these
other chemicals from our spectrophotometer reading we should get clearer results. The
blank is a way of doing exactly that. The blanks in this experiment include all of the
reagents except the enzyme.
The blank often serves another purpose in experiments like this one. Sometimes
there may be a color change in the experimental tubes due to oxidation of the
substrate or other non-enzymatic factors. Since the blanks also contain the substrate,
any oxidation will occur in the blanks as well. Therefore if you “zero” the Spec 20 with
the blank before measuring the absorbance of your experimental tube, any absorption
due to oxidation will not show up in the spectrophotometer reading of the experimental
tube. See Table 1 for the make-up of the blanks but do not make them until instructed.
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A control is most often used to make sure that the reaction is working correctly.
Most commonly there will be a positive control and one or several negative controls.
The positive control will contain all of the reagents necessary to make the reaction
work. The negative control will contain all of the reagents necessary to make the
reaction work except one. The purpose of the positive control is to make sure the
reaction is working properly so that the reaction can be studied effectively. The
purpose of the negative control(s) is to make sure that the reaction does not proceed
when one of the key reagents is missing (as sometimes happens in some sensitive
assays).
Now let’s see if you can apply your knowledge. Do any of the blanks in this
experiment also qualify as a control? Want a hint? Think about the contents of the
blanks (see Table 1) and what controls are used for. Now, if you said that the blanks
can be used as negative controls you are right! Remember that a negative control is
used to make sure that the reaction does not proceed forward without the proper
ingredients and the blanks in this experiment could be used that way. But you will not
use the blanks in that way. You will use the blanks as blanks by using them to “zero”
the Spec 20 before measuring the absorbance of the experimental cuvettes. This
makes the experiment much simpler than if you used the blanks as negative controls.
Do any of the blanks qualify as positive controls? The answer is no, but in this
experiment a positive control is not really necessary because it will be very obvious if
the reaction doesn’t work. However if the experimental mixture is very complex or if
you were doing experiments with enzyme inhibitors, positive controls would be
necessary. As experiments get more involved, the proper use of controls is often
critical to the success of an experiment.
Turn on the Spectrophotometer and set the wavelength to 410nm. If you do not
remember how to use the Spectrophotometer, refer back to the spectrophotometry lab
exercise.
The product of this reaction is a hazardous material. If you get any of the
reaction mixture on your skin, wash off immediately and notify your instructor.
Make the blank cuvette first. But before you make them, note several things:
? Place the buffer in the cuvettes first. Then add deionized water and then the
enzyme to any cuvettes that require them. Do not add the substrate until you are
ready to start the experiment. The addition of substrate allows the reaction to
start. In fact, it is a good idea not to add anything beyond the buffer and any
water to any cuvette until you are ready to start the experiment. This limits any
non-specific reacting or oxidation to a minimum.
? Experiments such as you are about to perform are powerful tools to study
enzymes. On the other hand, they mean very little if they are performed without
precision. It is important that the correct reagent in the correct amount is added
to the correct test cuvette.
? Since adding the substrate to the enzyme starts the reaction, this poses a little
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problem. For you to be able to compare the results of any experimental cuvette.
with the results of any other cuvette, the length of time between
Spectrophotometer readings must be the same. It is impossible to add substrate
to all cuvettes at exactly the same time and it is impossible to take
spectrophotometer readings of all of the cuvettes at the same time. For that
reason it is necessary to devise a method for substrate addition and taking
spectrophotometer readings that takes this problem into account.
* Devise a method for taking Spectrophotometer readings that takes into account these
problems and show your instructor or lab assistant for a verification of your method
before you start
Get 10 test cuvettes and a rack. Label 5 test cuvettes 1B, 2B, 3B, 4B, 5B. Place
the required reagents in the cuvettes in the order listed in Table 1 above. Do not add
the substrate yet. Use one pipet for each reagent. It is best to label a pipet for use
with one reagent. If you don’t contaminate the pipet, you can keep using it for that
reagent. (However, if the reagent you are using is sterile or you contaminate the pipet
by touching it to a different reagent, you must use a new, sterile pipet. Note that we are
not using any sterile reagents in this experiment)
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Table 1: Blanks for Part I
Reagent
(in ml)
Cuvette
1B
Cuvette
2B
Cuvette
3B
Cuvette
4B
Cuvette
5B
Buffer 1.65 1.60 1.50 1.30 0.90
DI Water 0.05 0.1 0.2 0.4 0.8
Substrate 1.3 1.3 1.3 1.3 1.3
Total 3 ml 3 ml 3 ml 3 ml 3 ml
Table 2 shows the contents of the experimental cuvettes. Label 5 cuvettes “1E, 2E, 3E,
4E, 5E”. Place the required reagents in the cuvettes in the order listed in Table 2
below. Do not add the substrate yet. Use one pipet for each reagent. These cuvettes
are paired with their blank cuvettes that you have already made. The pairing works like
this: 1E is the experimental cuvette and 1B is its blank. 2E is paired with 2B, and so on.
Table 2: Experimental cuvettes for Part I
Reagent
(in ml)
Cuvette
1E
Cuvette
2E
Cuvette
3E
Cuvette
4E
Cuvette
5E
Buffer 1.65 1.60 1.50 1.30 0.90
Enzyme 0.05 0.1 0.2 0.4 0.8
Substrate 1.3 1.3 1.3 1.3 1.3
Total 3 ml 3 ml 3 ml 3 ml 3 ml
Add substrate to the experimental cuvette and blank using the method that you
have devised for dealing with the timing problem. If the volumes vary between
cuvettes, then you have made a mistake in pipeting.
Remember to mix your cuvettes immediately after adding the substrate. Mixing
can be done by vortex or by covering the cuvette. with Parafilm™ and inverting.
Using the correct blank, set the range of the Spectrophotometer using the
procedure that you learned in the Spectrophotometry Lab Exercise. Remember: before
each measurement of an experimental cuvette, “zero” the spec with the specific blank
paired to that experimental cuvette.
Start taking measurements immediately and continue every 30 seconds for 3.5
minutes. Record your absorbance readings in Table 3.
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Table 3: Experimental Cuvette Absorbance Readings for Part I
Absorbance Readings at Different Times
cuvette 0
sec
30
sec
60
sec
90
sec
120
sec
150
sec
180
sec
210
sec
240
sec
270
sec
1E
2E
3E
4E
5E
*When you have take all of your readings, remember to dispose of all solutions in
the hazardous waste container. Do not dump the solutions down the drain. Clean
up.
It is time to analyze your data. Using a sheet of graph paper, (or using Excel™)
make a line (“connect the dots”) graph of Absorbance vs. time. Each experimental
cuvette should be represented on this graph. This graph shows the enzymatic activity
of the enzyme under “normal” conditions at different enzyme concentrations.
This experiment determines how much enzyme to use in Part II/ The amount to
be used in the following experiments is that amount that showed a linear absorbance
change for 3 min. on your graph. Show your graph to the instructor or lab assistant to
verify which amount of enzyme is correct to use in the second set of experiments.
Place your results on the board and write the correct volume of enzyme to use in this
space:_____ml
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Questions for Part I:
1. What is the purpose of blank and control cuvettes?
2. Why do we have a blank for each experimental cuvette and not just one blank
for the whole experiment?
3. To make this experiment simpler, the controls were not included. If you were to
include positive and negative controls what would they contain and why?
4. Theoretically, given enough time, will the final absorbance of cuvettes 1E, 2E,
3E, 4E and 5E be the same? Why or why not?
5. Since the original enzyme stock solution has a concentration of 0.1g/l, what final
concentration of enzyme gave the optimal results and should be used in the rest
of the experiments?
6. In the experimental cuvettes with a high enzyme concentration, the absorbance
will likely flatten out near the end of the experiment. Assuming that that alkaline
phosphatase is not a reversible enzyme, what do you believe is the reason for
the flattening out of the curve?
7. Why did you carry out Part I? Why not just use a high concentration of the
enzyme in Part II and not bother with Part I?
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Part II: How do environmental factors affect the activity of alkaline phosphatase?
Your body and all other living things maintain an internal environment that is
very stable in pH, solute concentration and in many cases temperature. The body uses
a lot of energy maintaining this “homeostatic” environment so it must be very important.
One reason homeostasis is important is that enzymes are very sensitive to
environmental changes. In these experiments you will demonstrate this.
There are three environmental conditions that the class will test for their ability to
affect enzyme activity. These are temperature, substrate concentration and pH. Your
group will be assigned one of these experiments to perform.
But before you start, note several things:
? If time permits, each group should run the experiment at least twice to verify the
results.
? Make sure that all conditions for the experiment are the same except for the
environmental factor to be studied.
? Use the correct blank(s) for the reasons described in Part I.
? Use the same basic experimental set up and procedures followed in Part I. If
you are unsure what this means ask your instructor or lab assistant.
? When you are finished with an experiment, clean up before you start another
experiment.
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Variable Temperature Experiment:
? All the experimental cuvettes will have the same composition. This will be the
same as the optimal enzyme concentration cuvette in Part I. Why?
? Use 5 experimental temperatures: ice bath, room temperature, 37oC bacterial
incubator or water bath, 50oC water bath, 70oC water bath).
? Fill in Table 4 with all of the contents of your experimental cuvettes (like Table 2)
and show it to the instructor or the lab assistant before mixing your cuvettes.
? Make sure you make the correct blank(s). Do you need 5 different blank
cuvettes or can one cuvette act as a blank? What will the composition be?
? At what temperature do you incubate the experimental cuvettes and the
blank(s)?
? Fill in Table 5 with the contents of the blank(s) for your experiment. Again, show
it to the instructor or lab assistant before starting to mix the cuvettes. Label and
make blank(s).
? Label and make up the 5 experimental cuvettes without the substrate and place
at the incubation temperature for at least 5 minutes before addition of the
substrate. This allows the reaction mixture to reach the reaction temperature
before the reaction starts.
? Using the same experimental procedures as before, add the substrate and take
absorbance readings every 30 seconds for 3.5 minutes and record in Table 6.
? When you have take all of your readings, remember to dispose of all solutions in
the hazardous waste container. Do not dump the solutions down the drain.
Clean up.
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Variable pH Experiment:
? All the experimental cuvettes will have the same composition except for one
important factor. They will have the same composition as the optimal enzyme
concentration cuvette in Part I except that each cuvette will have a buffer of a
different pH.
? Fill in Table 4 with all of the contents of the experimental cuvettes (like Table 2)
and show it to the instructor or the lab assistant before mixing up your cuvettes.
? Make sure you make the correct blank(s). Do you need 5 different blank
cuvettes or can one cuvette act as a blank? What will the composition be?
? At what temperature do you incubate the experimental cuvettes and the
blank(s)?
? Fill in Table 5 with the contents of blank cuvette(s) for your experiment. Again,
show it to the instructor or lab assistant before starting to mix the cuvettes. Label
and make blank(s).
? Label and make 5 experimental cuvettes without the substrate. Using the same
experimental procedures as before, add the substrate and take absorbance
readings every 30 seconds for 3.5 minutes and record in Table 6.
? When you have take all of your readings, remember to dispose of all solutions in
the hazardous waste container. Do not dump the solutions down the drain.
Clean up.
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Variable Substrate Concentration Experiment:
? In varying the substrate concentration (initial conc. of substrate is 800µg/ml), the
volume of the substrate will change. Make the volume of substrate in the
experimental cuvettes between 0.1ml and 1.0ml to avoid making the buffer too
dilute. The volume of the substrate changes but the total volume in each
cuvette. must stay the same. How do you solve this problem?
? Fill in Table 4 with all of the contents of the experimental cuvettes (like Table 2)
and show it to the instructor or the lab assistant before mixing up your cuvettes.
? Make sure you make the correct blank(s). Do you need 5 different blank
cuvettes or can one cuvette act as a blank? What will the composition be?
? At what temperature do you incubate the experimental cuvettes and the
blank(s)?
? Fill in Table 5 with the contents of blank(s) for your experiment. Again, show it to
the instructor or lab assistant before starting to mix the cuvettes. Label and
make blank(s)
? Label and make 5 experimental cuvettes without the substrate. Using the same
experimental procedures as before, add the substrate and take absorbance
readings every 30 seconds for 3.5 minutes and record in Table 6.
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Table 4: Blanks for Part II
Reagent
(in ml)
Cuvette 1B Cuvette 2B Cuvette 3B Cuvette 4B Cuvette 5B
Buffer
Enzyme
Substrate
Total 3 ml 3 ml 3 ml 3 ml 3 ml
Table 5: Experimental Cuvettes for Part II
Reagent
(in ml)
Cuvette 1E Cuvette 2E Cuvette 3E Cuvette 4E Cuvette 5E
Buffer
Enzyme
Substrate
Total 3 ml 3 ml 3 ml 3 ml 3 ml
Table 6: Experimental Cuvettes Absorbance Readings for Part II
Absorbance Readings at Different Times
Cuvette 0
sec
30
sec
60
sec
90
sec
120
sec
150
sec
180
sec
210
sec
240
sec
270
sec
1E
2E
3E
4E
5E
When you have taken all of your readings, remember to dispose of all solutions in the
hazardous waste container. Do not dump the solutions down the drain. Clean up.
15
Questions for Part II:
State the composition of your blank(s), and state specifically why the blank(s)
was/were needed.
For all experimental cuvettes, make a line (“connect the dots”) graph of absorbance vs.
time using Excel™.
Compare the graphs and the data you generated and answer these questions:
1. Describe any difference in the 5 graphed lines of your experimental cuvettes.
How do you explain any difference based on the different compositions of the
cuvettes?
2. If given enough time, will all of the experimental cuvettes reach the same
absorbance? Why?
3. Calculate the slope of the line (at a segment where the line is straight) for each
experimental cuvette. Place in Table 7. Remember that:
m= y2
– y1
/ t2
– t1
where m= slope
y1
= data point at time 1
y2
= data point at time 2
t1
= time 1
t2
= time 2.
In reality, the slope of a graph of a reaction equals the velocity of the reaction (i.e. how
fast the reaction is occurring)
Table 7: Slope values for Part II
Cuvette Slope Values
1E
2E
3E
4E
5E
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4. Now plot the slope value of each experimental cuvette vs. the variable being
studied. For example, if you studied the effect of pH change on the reaction,
plot the slope value of the experimental cuvette vs. the pH of that same
cuvette. Do this for each experimental cuvette and plot as a line graph with a
best fit line or a line that connects the data points (use the method that best
represents the data).
5. Looking at this graph, make a general statement about how the reaction rate
was changed by changing the variable that you studied.
*What you have generated is a graph showing how the rate of the reaction
accelerates or decelerates as you change the conditions of the reaction.
This lab exercise was developed in part with the support of National Science Foundation (Division of
Undergraduate Education) grant # DUE 9552290 and California Community College Chancellor’s Office
(Curriculum and Instructional Resources Division, Special Projects) grant # FII 95-621-001.
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