Part 1 - Get a Lab Appointment and Install Software:
Set up an Account on the Scheduler (FIRST TIME USING NANSLO):
Find the email from your instructor with the URL (link) to sign up at the scheduler.
Set up your scheduling system account and schedule your lab appointment.
NOTE: You cannot make an appointment until two weeks prior to the start date of this lab assignment.
You can get your username and password from your email to schedule within this time frame.
Install the Citrix software: – go to http://receiver.citrix.com and click
download > accept > run > install (FIRST TIME USING NANSLO).
You only have to do this ONCE. Do NOT open it after installing. It will work automatically when you go
to your lab. (more info at
http://www.wiche.edu/info/nanslo/creative_science/Installing_Citrix_Receiver_Program.pdf)
Scheduling Additional Lab Appointments:
Get your scheduler account username and password from your email.
Go to the URL (link) given to you by your instructor and set up your appointment.
(more info at http://www.wiche.edu/nanslo/creative-science-solutions/students-scheduling-labs)
Changing Your Scheduled Lab Appointment:
Get your scheduler account username and password from your email. Go to http://scheduler.nanslo.org
and select the “I am a student” button. Log in to go to the student dashboard and modify your
appointment time. (more info at http://www.wiche.edu/nanslo/creative-science-solutions/studentsscheduling-labs)
Part 2 – Before Lab Day:
Read your lab experiment background and procedure below, pages 1-14.
Submit your completed Pre-Lab Questions (pages 8-9) per your faculty’s instructions.
Watch the Spectrophotometer Control Panel Video Tutorial
http://www.wiche.edu/nanslo/lab-tutorials#beerlambert
Part 3 – Lab Day
Log in to your lab session – 2 options:
1)Retrieve your email from the scheduler with your appointment info or
2) Log in to the student dashboard and join your session by going to http://scheduler.nanslo.org
NOTE: You cannot log in to your session before the date and start time of your appointment. Use
Internet Explorer or Firefox.
Click on the yellow button on the bottom of the screen and follow the instructions to talk to your lab
partners and the lab tech.
Remote Lab Activity
SUBJECT SEMESTER: ____________
TITLE OF LAB: Enzyme
Lab format: This lab is a remote lab activity.
Relationship to theory (if appropriate): In this lab you will be examining the underlying
processes of enzyme action.
Instructions for Instructors: This protocol is written under an open source CC BY license. You
may use the procedure as is or modify as necessary for your class. Be sure to let your students
know if they should complete optional exercises in this lab procedure as lab technicians will not
know if you want your students to complete optional exercises.
Instructions for Students: Read the complete laboratory procedure before coming to lab.
Under the experimental sections, complete all pre-lab materials before logging on to the
remote lab. Complete data collection sections during your online period, and answer questions
in analysis sections after your online period. Your instructor will let you know if you are
required to complete any optional exercises in this lab.
Remote Resources: Primary – Spectrophotometer, Secondary - Enzyme.
CONTENTS FOR THIS NANSLO LAB ACTIVITY:
Learning Objectives........................................................................................................ 2
Background Information ............................................................................................... 2-8
Pre-lab Exercise 1: The Effect of Substrate Concentration on Enzyme Activity ........... 8-9
Pre-lab Exercise 2: The Effect of Temperature on Enzyme Activity ............................ 9
Equipment ..................................................................................................................... 9
Preparing for this NANSLO Lab Activity ......................................................................... 10
Experimental Procedure ............................................................................................... 10
Exercise 1: The Effect of Substrate Concentration on Enzyme Activity........................ 10-12
Exercise 2: The Effect of Temperature on Enzyme Activity ......................................... 12-13
Summary Questions ...................................................................................................... 14
Creative Commons Licensing ........................................................................................ 15
U.S. Department of Labor Information ......................................................................... 15
1|Page
Last Updated May 27, 2015
LEARNING OBJECTIVES:
After completing this laboratory experiment, you should be able to do the following things:
1.
2.
3.
4.
5.
6.
Define enzymes; know their functions and their characteristics.
Understand how enzyme activity can be affected by certain variables.
Observe and explain enzyme activity by means of a colorimetric enzyme reaction.
Use quantitative data to create a graph.
Determine the effect of temperature on enzymatic activity.
Determine the effect of substrate concentration on enzymatic activity.
BACKGROUND INFORMATION:
Living organisms contain enzymes, which are biological catalysts used to speed up chemical
reactions. Enzymes are vital to any living organisms; without their actions, chemical reactions
would not occur rapidly to be able to support life.
To get a better idea of why this is important think about the process of eating a meal. We know
that in a healthy diet we gain important nutrients. Nutrients are the molecules necessary for
organisms to grow, reproduce and repair themselves. The breakdown of these nutrients
provides energy and building blocks for living organisms via chemical reactions that will in turn
result in growth, reproduction and repair.
With the exception of one enzyme (the ribonuclease), all enzymes are protein molecules with
specific functions and characteristics.
First, the basic function of any enzyme is to increase the rate of a reaction. Most cellular
reactions occur about a million times faster than they would in the absence of an enzyme.
Second, most enzymes act specifically with only one reactant (called a substrate) to produce
products.1 So how does an enzyme work? An enzyme works by lowering the amount of energy
it takes to produce a reaction, this energy is called Activation Energy – the energy required to
break existing bonds to start a chemical reaction. For example to get a rock rolling down a hill, it
needs a push. That push is the activation energy. A Catalyst is something that acts to lower the
activation energy of a reaction. They reduce the “nudge” needed to get the rock rolling down
the hill. Additionally, an enzyme is not used up in the reaction; it can be reused when the
reaction is complete. Lastly, like all other proteins in a living cell, enzymes are produced from
genes in the organism’s DNA.
In summary, enzymes are protein biological catalysts used by cells to drive chemical reactions.
They are reusable and are produced under the direct control of an organism’s genetic material.
They work by binding to a specific molecule and putting stress on the bonds of that molecule so
the reaction is more likely to occur. If it were not for enzymes it may take weeks for our bodies
to break food down.
2|Page
Last Updated May 27, 2015
How Enzymes Speed Chemical Reaction Rates: A protein based enzyme is made up of stands of
amino acid chains called polypeptides. In the process of folding these poly peptides together,
folds and groves are created. In this video https://www.youtube.com/watch?v=yZ2aY5lxEGE 2
you can get an idea of how a protein is folded to create a 3 dimensional shape. While the video
is focused on proteins it is helpful to recall that the enzymes we are working with are proteins.
In an enzyme the groves created are called active sites then can react with other molecules by
physically fitting together with a molecule. The molecule to which an enzyme acts upon is the
substrate or reactant. When the enzyme attaches itself to the substrate molecule, a new
temporary molecule is formed the enzyme-substrate complex or ESC. While in the enzyme
substrate complex the molecules are in close proximity and the bonds are more easily altered;
as a result the activation energy is less than it would be without the enzyme. Figure 1 below
shows the breakdown of a large molecule into two smaller molecules with the use of an
enzyme. Enzyme activity can be measured in the amount of product formed also known as the
turnover rate.
Figure 1: Formation of an Enzyme Substrate Complex and Breakdown of the Substrate
Enzyme Helpers: Some enzymes use additional chemical components called coenzymes to aid in
lowering the activation energy; they are non-protein organic molecules. Many of the
coenzymes cannot be made by the organism, therefore must be obtained from food or are
made from vitamins. If the molecule is not organic it is called a cofactor. These cofactors
temporarily attach to the enzyme and work with the protein catalyst to speed up a reaction.
How the Environment Affects Enzyme Action:
1. Temperature has two effects on enzymes:
a. It can change the rate of molecular motion; and
b. It can cause changes in the shape of enzyme.
3|Page
Last Updated May 27, 2015
Figure 2: Enzymes and temperature
Optimum temperature is the temperature at which the rate of the formation of the
enzyme-substrate complex is fastest or the temperature at which the product is
formed most rapidly. When a temperature is raised above the optimum, the
enzyme changes its shape so that it can no longer form the enzyme-substrate
complex. A denatured enzyme is an enzyme whose protein structure has been
permanently changed so that is has lost its original biochemical properties and
unable to bond properly with the substrate.
2. The number of hydrogen ions dictates whether a pH will be acidic or basic. The amount
of hydrogen ions in an environment is very important in determining the shapes of
protein molecules; too many or too few H ions can alter the shape of the enzyme. Each
enzyme has its own optimum pH where the substrates will fit; most enzymes work best
in a pH at or near 7 (neutral).
Figure 3: Enzymes and pH
3. Enzyme-Substrate Concentration the concentration of the substrates, enzymes and
products influences the rates of enzymatic reactions. If the enzyme concentration
increases, the amount of product formed increases in a specific time; if the enzyme
numbers are decreased, the amount of product formed decreases. If the substrate is
present in a low concentration, enzyme activity will also be impacted. Products cannot
4|Page
Last Updated May 27, 2015
be formed until there is enough substrate and enough enzymes.
Figure 4: Enzymes and the Effect of Concentration
Cellular-Controlling Processes and Enzymes: Control processes are mechanisms that ensure that
an organism will carry out metabolic activities in the proper sequence (coordination) and at the
proper rate (regulation). Coordination is when the reactions take place in a specific order.
Regulation is how a cell controls the amount of chemical product produced. Below are a few of
the ways this can happen.
1. Gene Regulation – the number and kind of enzymes produced are regulated by the cell’s
genes. Gene regulator proteins – are chemical messengers that inform the genes as to
whether they should turn enzyme production on or off. There are two types a) generepressor proteins decrease protein production, b) gene-activator proteins increase
protein production.
2. Feedback mechanisms – is another method to control the synthesis of molecules.
Negative Feedback - As the numbers of products increase product molecules feedback
to a previous reaction point in the reaction chain and have a negative effect on the
enzyme controlling that reaction, thus decreasing the concentration. That is they inhibit
or prevent that enzyme from performing at its best. A good way to think of this is to
compare it to how a furnace works. If the temperature drops below the set temperature
the furnace kicks on and brings it back to the set temperature. An example of this in a
living system is regulation of blood glucose levels.
Positive Feedback- works in the opposite manner of negative feedback systems. As the
product of an enzyme reaction increases it acts to amplify the system. So in the example
of the furnace as the temperature increases to the set point on the thermostat the
thermostat would respond by keeping the furnace going thus increasing the
temperature even more. A biological example of this is the process of labor for child
birth.
5|Page
Last Updated May 27, 2015
3. Inhibition – occurs when the operation of enzymes is influenced by the presence of
other molecules. An inhibitor is a molecule that interferes with an enzymes ability to
form an enzyme-substrate complex; in doing so it removes a specific enzyme as a
functioning part of the cell resulting in no product formation.
A competitive inhibitor is any compound which closely resembles the substrate. This could be
having a similar chemical composition or having a similar shape. As a result the inhibitor
competes for the same active site as the substrate molecule. Competitive inhibition can be
reversible if sufficient substrate molecules are available to ultimately displace the inhibitor.
A noncompetitive inhibitor is a substance that binds with the enzyme, but not necessarily at
the active site. The noncompetitive inhibitor can bind to the enzyme and cause a shape change
in the active site. As a result the substrate can no longer bind with the enzyme to cause a
reaction. A non-competitive inhibitor is usually reversible, but is not influenced by
concentrations of the substrate.
Enzyme inhibition can be used to control diseases caused by bacteria by having antibiotics’
active ingredients act as the bacteria’s substrate, thus preventing the bacteria from producing
the enzymes they need to function. Some bacteria can survive and become resistant to certain
antibiotics. This can occur in 4 ways: The target is what the enzyme works on.
1.
2.
3.
4.
They can stop producing the molecule that is the target of the drug.
They can become impermeable to the drug.
They can modify the target.
They can release enzymes that inactivate the antibiotic.
This laboratory activity will focus on how different temperatures and different substrate
concentrations affect the rate of an enzyme reaction. We will be using the enzyme
glucose oxidase.
Glucose oxidase
𝛽 − 𝐷 − 𝐺𝑙𝑢𝑐𝑜𝑠𝑒 + 𝑂22 + 𝐻2 𝑂 → 𝐷 − 𝐺𝑙𝑢𝑐𝑜𝑛𝑖𝑐 𝐴𝑐𝑖𝑑 + 𝐻2 𝑂2
Peroxidase
𝐻2 𝑂2 + 𝑃ℎ𝑒𝑛𝑜𝑙 + 𝑅𝑒𝑑𝑢𝑐𝑒𝑑 𝐷𝑦𝑒 → 𝑂𝑥𝑖𝑑𝑖𝑧𝑒𝑑 𝐷𝑦𝑒 + 𝐻2 𝑂
The reaction of the enzyme glucose oxidase with the β-D Glucose produces D-Gluconic acid
and hydrogen peroxide (H2O2). The hydrogen peroxide then reacts with a color producing
chemical to produce a color. The amount of product formed can be related to the intensity of
color produced; which is then directly related to the amount of glucose used initially. Notice
6|Page
Last Updated May 27, 2015
that the greater the color intensity, the higher the concentration of the product. You will use a
spectrophotometer to measure the absorbance. The color change we are looking for will be a
pinkish color.
There are several relationships that enable us to measure enzyme activity using
spectrophotometry. Since the rate of product formation during an enzyme-catalyzed reaction
correlates with enzyme activity, and since the rate that absorbance changes during the reaction
is proportional to the rate of product formation, then the rate that absorbance changes is
proportional to enzymes activity.
Figure 5: Spectrophotometer
http://www.altechna.com/product_details.php?id=1175 (Accessed 8/1/2014)
How a Spectrophotometer works: In Figure 5 you can see the basic components of a
spectrophotometer. In this simple diagram you can see the basics of how the
spectrophotometer works. The light source will provide all wavelengths of visible light and
wavelengths in the ultraviolet and infrared range as well. The light enters the
spectrophotometer through a fiber optic port and some filters (1, 2 and 3 in the figure), and hits
a series of mirrors and diffraction gratings (4, 5 and 6), which act to separate the light into its
wavelengths. The separated wavelengths are then focused through some lenses (7) onto the
detector array (8) simultaneously. The detector then sends its data to some other electronics
(9 and 10), which turn the signal into the spectrum graph that you see on your computer.
In experiments using a spectrophotometer a blank is used in experiments. We use blanks for
several reasons. A spectrophotometer is used to measure the formation of a product as a
result of a reaction. However before measuring the absorption of the product you need to
know what the absorbance of the reactants are. For example in the experimental reaction we
will be working with the enzyme glucose oxidase, the substrate β-D Glucose which produces
D-Gluconic acid and hydrogen peroxide (H2O2). The hydrogen peroxide then reacts with a color
7|Page
Last Updated May 27, 2015
producing chemical to produce a color. The colored product of this reaction absorbs at 441.4
nm.. Remember what you are looking for are the 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, or reference sample, in this experiment include all of the reagents
except the colored product produced by the enzyme.
Sources:
1. http://www.elmhurst.edu/~chm/vchembook/570enzymes.html
2. https://www.youtube.com/watch?v=yZ2aY5lxEGE
PRE-LAB EXERCISE 1: The Effect of Substrate Concentration on Enzyme Activity
In this experiment, you will be exploring the effect of varying substrate concentration on the
rate of the enzyme reaction. In this exercise you will collect absorbance data and then create a
graph. Your graph can be used to calculate the initial reaction rate.
As with all chemical reactions whether they are mediated by biological enzymes or not, the
concentration of your reactants affect the reaction. In this exercise, you will be examining the
effect of glucose concentration on the rate of the reaction. You will want to keep the
concentration of the enzyme constant while varying the concentration of glucose. Enzyme
activity is measured in units. In this activity, the stock concentration of enzyme solution is 12.5
u/mL. We will keep the final concentration of the enzyme at 4.2 u/mL. Each cuvette used in
this experiment will contain 1.0 mL of the enzyme solution, and you will add glucose solution
and distilled water to reach a total volume of 3.0 mL for each test. The glucose stock solution
will have a concentration of 0.10 mg/mL. You will have a total of 5 cuvettes to work with in this
activity. In the first 3 cuvettes, you need to add glucose stock solution and water to create a
solution that has between 0.003 mg/mL and 0.70 mg/mL of glucose in the solutions. After
observing the behavior of the three solutions, you will decide which concentration to use for
the last two cuvettes where you will investigate the effect of temperature on the reaction.
Pre-lab Questions:
1. To prepare for the lab activity, create a table and determine the volumes of distilled
water and glucose solution you are going to add in the first three cuvettes. Record both
volumes in your table.
2. Do you think you will see a pattern in the rate of turnover as the concentration of
glucose changes? Explain your answer.
3. Hypothesis/Prediction – Set this up as an - if…..then…… statement. For example: If heat
is applied to particles in random motion then observable differences will be seen in the
absorption at the different temperatures. This example is meant to be very general;
8|Page
Last Updated May 27, 2015
your job is to use your answer to question #2 and make it into a more specific if-then
statement based on your understanding prior to conducting the experiment.
PRE-LAB EXERCISE 2: The Effect of Temperature on Enzyme Activity
From the background we know that temperature can have two effects on enzymes, there can
be a change in the rate of molecular activity or there can be a change in the shape of the
enzyme molecule. Both of these will have an impact on turnover rate. Enzyme activity is usually
expressed in terms of the rate of the reaction catalyzed by the enzyme. The rate is defined as
the amount of substrate transformed, or the amount of product formed, per unit of time. We
will measure the change in the absorption spectrum at 441.4 nm as the reaction proceeds.
When the data is graphed, you can determine the slope of the line which will give you the
reaction rate.
Pre-lab Questions:
1. Do you think you will see a pattern in the rate of turnover as temperature changes?
Explain your answer.
2. What color do you predict the product of the reaction will be? Defend your answer.
3. What is the enzyme used in this experiment?
4. What is the substrate used in this experiment?
5. What solutions should be in the blank?
6. Hypothesis/Prediction – Set this up as an - if…..then…… statement. For example: If heat
is applied to particles in random motion then observable differences will be seen in the
absorption at the different temperatures. This example is meant to be very general;
your job is to use your answer to question # 1 and make it into a more specific if-then
statement based on your understanding prior to conduction the experiment
EQUIPMENT:



Paper
Pencil/pen
Computer with spreadsheet program and Internet access (for the remote laboratory and
for data analysis)
9|Page
Last Updated May 27, 2015
PREPARING FOR THIS NANSLO LAB ACTIVITY:
Read and understand the information below before you proceed with the lab!
Scheduling an Appointment Using the NANSLO Scheduling System
Your instructor has reserved a block of time through the NANSLO Scheduling System for you to
complete this activity. For more information on how to set up a time to access this NANSLO lab
activity, see www.wiche.edu/nanslo/scheduling-software.
Students Accessing a NANSLO Lab Activity for the First Time
For those accessing a NANSLO laboratory for the first time, you may need to install software on
your computer to access the NANSLO lab activity. Use this link for detailed instructions on
steps to complete prior to accessing your assigned NANSLO lab activity –
www.wiche.edu/nanslo/lab-tutorials.
Video Tutorial for RWSL: A short video demonstrating how to use the Remote Web-based
Science Lab (RWSL) control panel for the air track can be viewed at
http://www.wiche.edu/nanslo/lab-tutorials#beerlambert.
NOTE: Disregard the conference number in this video tutorial.
AS SOON AS YOU CONNECT TO THE RWSL CONTROL PANEL: Click on the yellow button at the
bottom of the screen (you may need to scroll down to see it). Follow the directions on the pop
up window to join the voice conference and talk to your group and the Lab Technician.
EXPERIMENTAL PROCEDURE:
Once you have logged on to the spectrophotometer you will perform the following laboratory
procedures.
EXERCISE 1: The Effect of Substrate Concentration on Enzyme Activity
As with all chemical reactions whether they are mediated by biological enzymes or not the
concentration of your reactants effects the reaction. In this exercise you will be examining the
effect of glucose concentration on the rate of the reaction. For this experiment, you will want
to keep the concentration of the enzyme constant while varying the concentration of glucose.
Enzyme activity is measured in units. In this activity, the stock concentration of enzyme
solution is 12.5u /mL. We will keep the final concentration of the enzyme at 4.2 u/mL. Each
cuvette used in this experiment will contain 1.0 mL of the enzyme solution , and you will add
glucose solution and distilled water to reach a total volume of 3.0 mL for each test. The glucose
10 | P a g e
Last Updated May 27, 2015
stock solution will have a concentration of 0.10 mg/mL. You will have a total of 5 cuvettes to
work with in this activity. In the first 3 cuvettes, you need to add glucose stock solution and
water to create a solution that has between 0.003 mg/mL and 0.070 mg/mL of glucose in the
solutions. After observing the behavior of the three solutions, you will decide which
concentration to use for the last two cuvettes, where you will investigate the effect of
temperature on the reaction.
Data Collection:
1. Click the Spectrometer tab and set # Spectra to Average to 20. This will give a clean
signal. Save the dark spectrum.
2. Turn on the spectrometer Light by clicking the Light button.
3. Click on the Qpod tab. Set the temperature to 370C and make sure the Stirring control is
set to On.
4. After the system indicates that it has reached the set temperature, wait two minutes to
allow everything to equilibrate.
5. Select cuvette 0 from the Cuvette Holder/Cuvette Select and Volume tab.
6. From the Spectrometer tab, save the Reference spectrum. Enable the cursor and place
it on 441.4 nm on the spectrum.
7. Click on the “Show Absorbance Spectrum” button to display the absorbance spectrum.
“Zoom Out” on the graph so you can see the features of the spectrum.
8. Click the Spectrometer/Value Log tab, set the total length of time to collect data to a
value between 3-5 minutes. Set the collections intervals to a value between 10-30
seconds.
9. From the Cuvette Holder/Cuvette Select and Volume tab, select cuvette 1.
10. Inject the distilled water and then the glucose solution. Start collecting data by clicking
Start on the Value Log tab.
11. Export Values Log to the clipboard and paste it into a spreadsheet.
12. After all group members have the Value Log data, click the Clear button to erase it.
13. Have another student in your group take control of the system.
14. Repeat steps 9-13 with cuvette 2 and your second concentration.
15. Repeat steps 9-13 with cuvette 3 and your third concentration.
Analysis:
1. Using the data exported from the remote lab, create a graph. On the graph you will
plot time as the independent variable and absorbance as the dependent variable. You
should have three different lines for the three different concentrations of glucose.
2. With your graphed data, calculate the slope of the line for the most linear portion of
the data collected at each concentration. Depending on how long you collected data
and how concentrated the glucose was, your graph may flatten out like the one shown
below. However, it is only the initial, relatively linear portion of the data that are useful
11 | P a g e
Last Updated May 27, 2015
for this purpose. In this graph, the useful data points are between 0.00 minutes and
2.00 minutes.
a.
Slope is calculated by first choosing two points on the graph then determining the
change in the horizontal points and the change in the vertical points. For example
if your coordinates on the graph at point 1 are 1(x axis) and 6 (y axis) and at point
2 they coordinates are 4(x axis) and 12 (y axis). So the vertical change is 12 - 6 and
the horizontal change is 4 – 1 or vertical change = 6 and horizontal change = 3.
Then to find the slope you would divide the vertical change by the horizontal
change. 6/3 for a slope of 2. Be sure to include the correct units for your data.
What information can you get from this graph?
3. In what way is the rate of the reaction dependent on the concentration of the
substrate? How would this change if we were testing the concentration of the enzyme?
4. In the experiments with a high substrate concentration, the absorbance will likely
flatten out near the end of the experiment. If the glucose oxidase is not a reversible
enzyme, what do you believe is the reason for the flattening out of the curve?
5. Based on the data you collected write a claim/evidence statement. In other words
make a claim based on what you learned in this experiment and back it up with the
data you collected.
Absorbance
Absorbance vs. Time - Glucose Oxidase
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0.00
1.00
2.00
3.00
4.00
5.00
6.00
Time (min)
EXERCISE 2: The Effect of Temperature on Enzyme Activity
You are measuring reaction rates (change in product concentration per unit time). Therefore, it
is important that you take your absorbance measurements at the precise time intervals. In
addition, you must keep in mind that even when you are taking your absorbance measurement
the reaction will still be proceeding, and more enzymatic product will be forming. Do not wait
12 | P a g e
Last Updated May 27, 2015
for the numbers to stop changing—they won’t! This also means that you should not “pause”
your timing of the reaction when you are taking your readings from the spectrophotometer.
Data Collection:
1. On the Spectrometer tab, ensure that the cursor is still enabled on the spectrum and is
on 441.4 nm and the absorbance spectrum is still being displayed.
2. On the Cuvette Holder/Temp Control tab, set the temperature to 10 0C and ensure that
the Stirring Control is still set to On.
3. After the system indicates that it has reached the set temperature, wait two minutes to
allow everything to equilibrate.
4. Select cuvette 4.
5. Click the Spectrometer/Value Log tab, and set the total length of time to collect data to
a value between 3-5 minutes. Set the collections interval to a value between
10-30 seconds.
6. Inject the distilled water and then the glucose solution and start recording data on the
Value Log tab as before.
7. Export Value Log to the clipboard and paste it into a spreadsheet.
8. After all team members have the Value Log data, click the Clear button to erase it.
9. Have another student on your team take control of the system.
10. Select cuvette 5 and repeat steps 6-9 at 70°C.
Analysis:
1. Using the data exported from the remote lab, create a graph. On the graph you will plot
time as the independent variable and absorbance as the dependent variable. You should
have two different lines for the two temperatures.
2. With your graphed data calculate the slope of the line for the initial, mostly linear
portion of the data collected at each temperature. What information can you get from
this graph?
3. Analyze the graph by relating the shape of the curve to an underlying mechanism that
might govern the phenomenon being studied. Discuss factors involved in enzyme action
that might cause this curve shape to be true.
4. On your time versus absorbance graph for each temperature extrapolate after 30
minutes what would the absorbance be at your highest temperature? Would the graph
continue to rise? Explain your reasoning.
5. How is the activity of the enzyme affected by temperature?
6. Based on the data you collected write a claim/evidence statement. In other words make
a claim based on what you learned in this experiment and back it up with the data you
collected.
13 | P a g e
Last Updated May 27, 2015
SUMMARY QUESTIONS
1. Why do you need to know the wavelength for maximal absorption first?
2. In these experiments is the blank the same thing as a control?
3. Explain what the absorbance measured in this activity will tell you about the activity of
the enzyme.
4. Predict what would happen if the enzyme was boiled for 30 minutes then reacted with
the substrate. Do some research to explain and defend your answer.
5. If given enough time, will all of the experimental tests reach the same absorbance?
Why?
6. If you vary pH, how do you think this will affect an enzyme? Why?
7. Explain the role of enzymes inside our body. Provide examples of enzymes.
Choose two of the following research questions to answer:
1. Research enzyme inhibition and write out a paragraph describing a method of testing
for enzyme inhibition.
2. Research and choose an enzyme related genetic disorder. Write a short summary of the
enzyme affected and how it impacts the body.
3. Research and explain how the three-dimensional shape of an enzyme is determined?
4. Lactose-intolerance is a very common condition worldwide. Using reliable scientific
resources research and write in a short paragraph about this condition.
5. Most viruses (with the exception of a few) lack basic metabolic enzymes but yet they
manage to infect our body and lead to serious diseases and infections. Discuss how they
are capable of doing all chemical reactions inside the host cells.
6. Imagine you are working in international research laboratory as a prominent research
scientist. Knowing that each enzyme has a set of optimal conditions (pH, temperature).
Describe how you will go about to find out the optimal conditions for a newly
discovered enzymes.
14 | P a g e
Last Updated May 27, 2015
For more information about NANSLO, visit www.wiche.edu/nanslo.
All material produced subject to:
Creative Commons Attribution 3.0 United States License 3
This product was funded by a grant awarded by the U.S.
Department of Labor’s Employment and Training Administration.
The product was created by the grantee and does not necessarily
reflect the official position of the U.S. Department of Labor. The
Department of Labor makes no guarantees, warranties, or
assurances of any kind, express or implied, with respect to such
information, including any information on linked sites and
including, but not limited to, accuracy of the information or its
completeness, timeliness, usefulness, adequacy, continued
availability, or ownership.
15 | P a g e
Last Updated May 27, 2015