BIO 122: Cells and Genetics

Recitation Week 1
Goals:
1. Learn about lab and recitation expectations
2. Learn about the SDH Activity Project
3. Ask questions about lecture material
Prior

to Recitation:
Read this entire packet about the SDH Activity Project
Come to recitation with any questions you might have
Complete the assignment on the last page of this handout. Your answers do not need to be
100% correct to earn full credit. Please just do your best to illustrate your current
understanding of the project. Answers to these questions will contribute to Week 1
Attendance for recitation. You will not earn Attendance credit for Week 1
Recitation unless you 1) attend Week 1 Recitation, and 2) bring the completed
questions at the end of this packet to Recitation in Week 1.

SDH Activity Project*

Project Goals
For this project, student lab groups will perform a project on the enzyme succinate
dehydrogenase (SDH). The goals of the project are as follows:
1. To understand the goal and process of cell fractionation and to gain practice with one
approach for fractionating cells,
2. To gain practice micropipetting,
3. To learn the rationale behind the Bradford assay and to practice conducting this assay,
4. To work with enzymes in the lab to gain a greater understanding of how enzymes work and
how they are affected by a variety of factors,
5. To generate a hypothesis based on information from the literature,
6. To test your hypothesis,
7. To interpret results and conclude as to whether your data support or refute your
hypothesis,
8. To generate graphs of scientific data and to practice graphing, and
9. To understand the electron transport chain and how it works coordinately with the
Krebs/TCA/citric acid cycle.
*This laboratory was adapted from a lab developed by Mary Lee S. Ledbetter, Department of Biology, College of the
Holy Cross, as part of a project for the American Society for Cell Biology Education Committee, 1992. Unless otherwise
indicated, all background material and project information was taken or adapted from this source.

Introduction to Enzymology, SDH, Cell Fractionation and the Bradford Assay

Enzymes**
**This section was adapted from a lab developed by Beth Nichols at Princeton University as part of the PEW
Undergraduate Laboratories in Biology Project.

Enzymes are biological catalysts that facilitate specific chemical reactions in living cells.
They are generally large proteins, or groups of proteins, each made up of several hundred amino
acids. They often contain a non-proteinaceous, prosthetic group, important for catalysis.
In an enzyme-catalyzed reaction, the substance to be acted upon, or substrate, binds to
the active site of the enzyme. The enzyme and substrate are held together in an enzymesubstrate complex by hydrophobic bonds, hydrogen bonds and ionic bonds. The enzyme then

facilitates the conversion of the substrate to product in a process that often requires several
chemical steps, and may involve covalent bonds. Finally, the product is released and the
enzyme is ready to form another enzyme-substrate complex. As a catalyst, the enzyme is not
used up as it carries out the reaction, but is repeatedly recycled. One enzyme molecule can
carry out thousands of reaction cycles every minute.
Each enzyme is specific for a certain reaction because its amino acid sequence is unique
enabling a unique three-dimensional structure. The active site also has a specific shape so that
only one or a few of the thousands of compounds present in the cell can productively interact
with it. Any substance that blocks or changes the shape of the active site will interfere with the
activity and efficiency of the enzyme. If these changes are significant, the enzyme may no longer
function. These changes to the enzyme may be permanent or irreversible. There are several
factors that are especially important in determining the enzymes shape, and these are closely
regulated both in the living organism and in laboratory experiments to give optimum or most
efficient enzyme activity:
1. SALT CONCENTRATION: If the salt concentration or ionic strength is very low or zero,
the charged amino acid side chains of the enzyme will stick together. The enzyme will
denature or unfold and form an inactive precipitate. If the salt concentration is very high,
there will be interference with the normal interaction and functioning of the charged
groups, which could cause precipitation of the enzyme. An intermediate salt concentration
such as that of blood (~0.9% or 150 mM NaCl) or cytoplasm is optimum for most enzymes.
2. pH: Enzyme amino acid side chains contain groups such as COOH and NH 2 that readily
gain or lose H+ ions (protons). As the pH is lowered, an enzyme will tend to gain H + ions,
and eventually enough side chains will be affected so that the enzymes shape is
disrupted. Likewise as the pH is raised, the enzyme will lose H + ions and eventually lose
its active shape. Many enzymes have an optimum in the neutral pH range (7.0) and are
denatured at either extremely high or low pH. Some enzymes, such as those which act in
the human stomach where the pH is very low, will have an appropriately low pH optimum.
A buffer is a compound that will gain or lose H + ions so that the pH of the solution changes
very little.
3. TEMPERATURE: Chemical reactions speed up as temperature is raised and more
molecules have sufficient kinetic energy to undergo the reaction. Since enzymes are
catalysts for chemical reaction, enzyme reactions also tend to proceed faster with
increasing temperature. However, above an optimum temperature the kinetic energy of
the system is so great that the structure of the enzyme starts to be disrupted. The positive
effect of speeding up the reaction is now more than offset by the negative effect of
denaturing more and more enzyme molecules. Many proteins are denatured by
temperatures around 40-50C, but some are still active at 70-80C.
4. SMALL MOLECULES: Many molecules other than the substrate may interact with an
enzyme. If such a molecule increases the rate of the reaction it is an activator, and if it
decreases the reaction rate it is an inhibitor. These molecules can regulate the rate of an
enzyme. A substance that unfolds the enzyme, such as an organic solvent or detergent,
will act as an inhibitor. Some inhibitors act by reducing the S-S- bridges that stabilize the
enzymes structure. Many inhibitors act by reacting with side chains in or near the active
site to change its shape, or block it. Others may damage or remove the prosthetic group.
Many well-known poisons, such as potassium cyanide and curare, are enzyme inhibitors
that interfere with the active site of critical enzymes.
Succinate Dehydrogenase

Succinate dehydrogenase or SDH is a mitochondrial enzyme that plays an important role

in the Krebs cycle (aka the TCA or Citric Acid Cycle) and in the electron transport chain which
function to generate ATP when oxygen is available. This enzyme, SDH, is the basis for this
project because: a) SDH activity can be easily assessed (assayed) in the lab using simple
techniques, b) there are safe modulators of SDH that we will study, and c) SDH is involved in
some of the pathways to be covered in lecture.
The chemical reaction carried out by SDH is shown in Figure 1. It catalyzes the oxidation
of succinate to fumarate. Flavine adenine dinucleotide (FAD) is a coenzyme covalently bound to
the SDH enzyme. As a part of this reaction, FAD receives electrons from succinate, which leads
to the generation of FADH2, a reduced form of FAD. In order for the enzyme to complete its
catalytic cycle so that it can react with the next succinate molecule, the electrons on reduced
FADH2 need to be passed down the electron transport chain, eventually to oxygen, thus leading
to the regeneration of oxidized FAD. As a result, SDH donates the electrons from FADH 2 to
ubiquinone (also called Coenzyme Q). From ubiquinone, the electrons are then donated to
complex III in the electron transport chain, then to complex IV, and ultimately onto oxygen, to
generate water.

Figure 1: Reaction carried out by SDH. Taken from Garrett & Grisham Biochemistry, 2 nd edition.
Assaying SDH Activity
Passage of electrons from FADH2 down the electron transport chain can be inhibited by
blocking the electron transport chain, allowing FADH 2 to accumulate. Artificial electron acceptors
can then be added to the system to draw electrons away from FADH 2, allowing oxidized FAD to be
regenerated. You will use an artificial electron acceptor called 2,6-dichlorophenol indophenols
(DCPIP), a dye whose absorbance differs depending upon its state of oxidation. In the oxidized
form, DCPIP absorbs at 600 nm forming a blue color, but it becomes colorless in its reduced form.
In the presence of SDH and substrate (succinate), FADH 2 will be formed. Inhibition of the electron
transport chain with azide allows FADH 2 to accumulate. As the electrons from reduced FADH 2 are
donated to DCPIP, the solution will become colorless. This color change can be quantitated over
time and will be used to quantitate SDH activity based on the generation of reduction of FAD by
SDH to FADH2 and the subsequent re-oxidation to FAD as the electrons are donated to DCPIP.
In this assay, you will add a variety of reagents to a tube or cuvette, which is ultimately
placed in a spectrophotometer. (Please see list of reagents below.) When doing an experiment,
it is essential to understand the role each reagent is playing in the reaction. When something
goes wrong with an experiment, having an understanding of the reagents can help you to
troubleshoot and improve upon the approach.
Reagents:
Potassium phosphate buffer to control pH of reaction, to maintain optimum range
for SDH
Azide blocks the electron transport chain to enable accumulation of FADH 2 based
on SDH activity
Buffered sucrose an osmotic buffer to keep mitochondria from bursting.
Mitochondrial extract source of SDH enyzme

DCPIP artificial electron acceptor to accept electrons from FADH 2 generated by

SDH; indicator dye, which absorbs at 600 nm (blue) when oxidized, but is colorless
when reduced.
Succinate substrate for SDH

The Spectrophotometer
You will use a spectrophotometer for a number of different assays during this project. A
spectrophotometer measures the ability of a substance to absorb a certain wavelength of light
(see Figure 2 for an example of how this works). It can separate wavelengths of light and send a
particular wavelength through a specimen. The photons that pass through the specimen are
detected and converted into electricity, which is reported by a galvanometer. The photons that
pass through the specimen were transmitted and not absorbed. In the case of the SDH assay,
DCPIP absorbs photons at 600 nm when oxidized, but when it is reduced, it no longer absorbs
photons of this wavelength, and thus those photons will be transmitted to the galvanometer.

Figure 2: How a spectrophotometer works. In this example, green light is being transmitted
through the sample of chlorophyll, and blue light is being absorbed by that pigment. Taken from
Campbell and Reece, 8th edition, Pearson/Benjamin Cummings
Cell Fractionation#
Material in this section was adapted from: Castle, J.D. (2001) Unit 3.1 Overview of Cell Fractionation. Current
Protocols in Cell Biology. Graham, J.M. (2001) Unit 3.4 Purification of a Crude Mitochondrial Fraction by Density
Gradient Centrifugation. And from Mary Lee S. Ledbetter, Department of Biology, College of the Holy Cross, as part of
a project for the American Society for Cell Biology Education Committee, 1992.
#

You will be using cow liver as your source of SDH. SDH is a mitochondrial enzyme that is
bound to the inner mitochondrial membrane. While most other tissues contain mitochondria,
liver contains a high concentration of mitochondria to accommodate its high demand for ATP.
While a crude extract of cow liver would certainly have SDH activity, you will fractionate
the bovine liver cells to obtain isolated mitochondria which will have a higher relative
concentration of SDH. Cell fractionation has been used by cell biologists for about 60 years. It
enabled biologists to identify various components of the cell and to isolate those cellular
components to assign and assay activities and to purify associated proteins.
Cell fractionation depends on differing physical properties of organelles, such as size,
shape, surface charge density and buoyant density, to allow for separation. In lab, we will be
using a centrifugation-based approach, a common cell fractionation procedure. Centrifugation

allows separation of cell components based on their size and density. Based on these properties,
particles will sediment at a specific velocity.
While there are ways to achieve nearly complete separation of particular cellular
components, these approaches are extremely time consuming, and thus not well adapted to an
undergraduate lab period. A quicker approach, though less complete, is to use differential
sedimentation. This approach allows for quicker isolation of mitochondria, which is all that is
needed for studying metabolic enzymes like SDH, which are present only in the mitochondrial
fraction. Note that this approach does not allow for complete separation of mitochondrial from
other cellular components, and therefore the mitochondrial fraction will actually be
contaminated with lysosomes, peroxisomes, Golgi membranes, and small amounts of ER
membranes. For our purposes, contamination with these organelles is not problematic, but if you
actually wanted to study the mitochondria in absence of these other cellular components, you
would want to use a more stringent procedure called density-gradient centrifugation.
For differential sedimentation, cellular components are suspended in a fluid (sucrose
solution), which is less dense than any of the cellular components. As you centrifuge the
sucrose/liver homogenate, the various cellular components will sediment to the bottom of the
tube, forming a pellet. Larger, denser particles will sediment quickly, and over time smaller, less
dense particles will begin to sediment. One way to isolate particular cellular components is to
stop the centrifugation process at some interval, remove the pellet, and continue to centrifuge
the material in the supernatant. This approach has been standardized to allow the isolation of
various cellular components.
One interesting point of note is that the SDH assay is actually used in some cases to
identify the mitochondrial fraction after a cell fractionation procedure, and to verify the purity of
each fraction. Since SDH is bound to the inner mitochondrial membrane, it should only be found
in the mitochondrial fraction. It is useful to conduct an SDH assay on all isolated fractions to look
at the relative SDH activity. The mitochondrial fraction should have high SDH activity, as SDH is
found solely in mitochondria. If another fraction (besides crude extract, which contains all
cellular components) also contains high SDH activity, that suggests that the fraction was
contaminated with mitochondria, and thus the fractionation was not successful. If a fraction of
interest (i.e. nuclear fraction) is contaminated with mitochondria, any discoveries you make
could be attributed to either the nuclear fraction or the contaminating mitochondrial fraction. In
our case, we are isolating the mitochondrial fraction so that we can study SDH activity
specifically. Ultimately, you are going to manipulate SDH activity using reagents, and making
predictions as to how those reagents might affect SDH activity based on your understanding of
the electron transport chain and the biological literature.
The Bradford Assay%
Material in this section was taken and adapted from: Bradford, M.M. (1976) A Rapid and Sensitive Method for the
Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Analytical Biochemistry
72: 248-54, as well as from materials written by Heather Blythe and Jessica Battisto
%

An important part of the experiments you will be conducting is to determine the protein
concentration of the mitochondrial fraction you prepare. The reason for this is that increasing
the concentration of enzyme present in a sample can increase the observed enzyme activity. As
you increase the amount of enzyme, you increase available sites for the substrate to bind and
form enzyme-substrate complex. As a result, you increase the amount of product you can
generate in a given time. Therefore, if you do not take protein concentration into account, you
might incorrectly conclude that SDH (or another enzyme you are studying) has higher activity

under a particular condition, when what you are actually seeing is increased activity due to an
increased concentration of SDH.
There are several assays that can be used to determine protein concentration. One
commonly used approach is the Bradford Assay. We have posted the original article from the
primary literature that describes this assay (Bradford, 1976). You should begin to familiarize
yourself with reading the primary literature, as this is something you will be asked to do
throughout your undergraduate career. The Bradford Assay is a reasonable way to assess
protein concentration, which is relatively quick and easy to perform.
This assay is based on the formation of a complex between a dye (Brilliant Blue G) and the
proteins in the solution. The protein/dye complex will absorb light at 595 nm, measurable with a
spectrophotometer. The amount of light absorbed is proportional to the amount of protein
present. The concentration range in which measurements can be appropriately made is between
0.1-1.4 mg/ml. To determine the amount of protein actually present in your sample, it is
essential to generate what is referred to as a standard curve. Basically, you use a standard or
a protein of known concentration (you will be using Bovine Serum Albumin, BSA), and generate
dilutions of this protein solution that contain known concentrations of that protein, and you run
these samples through the Bradford Assay. The absorbance values for these standards can be
plotted versus the concentration of proteins in these samples to generate the standard curve.
The absorbance values of the unknown protein samples can then be compared with the
standards to determine the approximate concentration of that unknown protein sample.
Ultimately, you will be measuring the protein concentration of the mitochondrial fraction
and crude extracts you generate via differential sedimentation. An important point of note is
that at no point in the procedure are you purifying the SDH enzyme, so protein levels that you
detect are not specifically SDH protein levels. Instead, you are measuring how much protein is in
the mitochondrial fraction versus the crude extract fraction. Though you do not know how much
SDH protein is specifically there, you are getting a general sense of how much protein is in your
sample, which allows you to make better relative comparisons between samples.
In order to make accurate comparisons between samples, it is useful to measure specific
activity. This is the amount of product formed by an enzyme in a given amount of time under
given conditions per mg of enzyme. Another way to define specific activity is the units of activity
(AU) per mg of enzyme. Note that this means that specific activity takes into account the
amount of enzyme present in the sample.
Introduction to Hypothesis Building, Interpreting Results, Presenting your Data
and
Arriving at a Conclusion
As a major in the sciences, you will likely be conducting hypothesis-driven experiments in
various lab courses throughout your undergraduate career. As a result, it is critically important
to introduce you to the process of generating your own hypotheses, interpreting the results you
obtain and presenting your data and arriving at a conclusion based on your data. Even if you are
not a science major and you are taking this course, as someone who is taking a course with a lab
component, it is critical for you to be exposed to the concept of hypothesis-driven science, and
for you to gain some experience with generating hypotheses and testing them. This project is
meant to provide a foundation in these skills.
In the first couple of weeks of this project, you will be learning techniques and gathering
the materials you need to do your experiment. For example, you will need to fractionate the
cells to obtain the mitochondrial fraction needed for the SDH assays, and you will need to
conduct the Bradford Assays to determine the concentration of protein in your samples.

However, ultimately, you will add a reagent assigned to your group to your SDH samples, and to
ask experimentally what the effect of that reagent will be compared to samples that have not
received the reagent.
Before you ask this question experimentally though, you will be asked to generate a
hypothesis as to what effect your assigned reagent will have on the activity of SDH. A
hypothesis is a testable prediction that is based on knowledge of the relevant variables. In order
to generate a useful hypothesis, it is absolutely critical that the hypothesis is well justified. It is
not appropriate to just make a guess as to the relationship between the variables; instead, the
hypothesis should be based on the previous work of others, or your own previous work. You will
be required to research your assigned reagent as a group and to develop a hypothesis based on
information in the biological literature. A rubric will be posted that explains what you need to do
for this assignment.
After you devise a hypothesis about the effects of your reagent on SDH activity, you will
have an opportunity to test your hypothesis. At this point, you will have an opportunity to
discover whether your hypothesis was correct or incorrect. A couple of important points of note:
You cannot prove a hypothesis in science. Instead, you should say that your results
were consistent with your hypothesis. You can, however, disprove a hypothesis.
If your results disprove your hypothesis, dont be disappointed! Some of the most exciting
discoveries have come from disproven hypotheses! If you find that your results disprove
your hypothesis, spend time thinking about why that might be. This is one of the most fun
parts of science figuring things out!
Once you have tested your hypothesis, you will have a variety of data that you will need to
assemble into graphs to present to others. An absolutely essential part of science is
communicating the information that youve learned to others. The more clearly you can do that,
the better. As part of this course, we will begin to teach you about some of the ways you can
present your data. Make sure that all graphs are clearly labeled and contain a figure legend.
The more clear and obvious your results are to an outside observer, the more likely it is that they
will get excited about your work!
Finally, you will communicate what you learned through a brief report that will include
background on your reagent, your hypothesis, a brief description of your results, and a
conclusion section. This will provide the foundation for future lab reports that you will submit in
this and other subsequent courses. Again, this is an essential step in the process of learning
about scientific written communication. A rubric will be posted that explains what you will need
to do for this assignment.
Assignment
You must bring your handwritten answers to these questions with you to recitation during
Week 1. If you do not bring this assignment with you to recitation during Week 1, your
participation grade will be docked.
1. Why are you studying SDH in this part of the course?

2. Why do you need to fractionate cells for the SDH assay? What is the goal of the cell
fractionation in this experiment?

3. What is the goal of the Bradford Assay, and why is it important to do as part of the SDH
Activity Project?

4. Can you prove a hypothesis in science?