Download Sickle Cell Workshop

Got Electrophoresis?
Funded by the National Science Foundation EPSCoR
(05-589)
Sickle-Cell Anemia
University of Southern Mississippi
Sherry Herron, Bridgette Davis, and Parker Nelson
May 7, 2008
Agenda
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A.M.
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8-9 Orientation, Survey, and Activity
Overview
9-12 Electrophoresis Chamber
Construction, Gel and Buffer preparation
P.M.
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12:30 Electrophoresis and Explanations
2-3 Post Activity Discussion and
Evaluation
Problem: The ubiquitous, yet obsolete and
oversimplified presentation of sickle cell disease
in biology textbooks.
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From newest
innovative NSFfunded high
school biology
textbook
~ 2 pages
Rationale for inclusion of Sickle
Cell Topics
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Research in cognition has demonstrated that learning
is most effective when topics are of human interest,
relate to one another (theme-oriented), and relate to
previously learned concepts (scaffolding). A focus on
sickle cell anemia satisfies these criteria and can
facilitate the teaching of a variety of biological,
chemical, social, medical, and ethical concepts. The
initial unit could serve as the basis for the
development of additional units designed for
evolution, genetics, medical school, biochemistry,
biotechnology, and bioethics college courses.
Statistics from the CBCF Health
Organisation, 2004
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Most cases occur in those with African ancestors in America
2 million people carry the sickle cell genetic trait in America
1 in 12 African Americans carry the sickle cell trait in America
About 91,000 people with sickle cell anemia ("Orphan Products: Hope
for People With Rare Diseases", By Carol Rados, FDA Consumer
magazine, November-December 2003 Issue)
1 in 500 African American births in America
1 in 1,000-1,400 Hispanic American births in America
Deaths from Sickle Cell Anemia: 501 deaths (NHLBI 1999)
Death rate extrapolations for USA for Sickle Cell Anemia: 500 per year,
41 per month, 9 per week, 1 per day, 0 per hour, 0 per minute, 0 per
second.
Statistics from the CDC
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Sickle cell disease affects millions of people throughout the world and is
particularly common among those whose ancestors come from sub-Saharan
Africa, Spanish-speaking regions in the Western Hemisphere (South America,
the Caribbean, and Central America), Saudi Arabia, India, and Mediterranean
countries such as Turkey, Greece, and Italy.
More than 70,000 people have sickle cell disease in the U.S.
Sickle cell disease occurs in 1 in every 500 African American births.
2 million people have sickle cell trait in the U.S.
1 in 12 African Americans has sickle cell trait in the U.S.
Did you know? Sickle cell disease occurs more often in people from parts of
the world where malaria is or was common. It is believed that people who carry
the sickle cell trait are less likely to catch malaria.
Sickle cell disease is a major public health concern. From 1989 through 1993,
there were an average of 75,000 hospitalizations due to SCD in the U.S., costing
~ $475 million.
Sickle cell anemia is the most common inherited blood disorder in the U.S.,
affecting about 72,000 Americans or 1 in 500 African Americans. (Source: Genes
and Disease by the National Center for Biotechnology)
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Malaria kills over 3000 children each day in Africa or
1 every 30 seconds
Malaria is the # 1 killer of children under 5 in subSaharan Africa
Malaria kills more than 1 million people each year
Young children and pregnant women are most
likely to become severely ill and die from malaria
Malaria was eradicated from the United States over
50 years ago, yet more than 40 percent of the
world's population is at risk
http://www.malarianomore.org/index.php
YOUR electrophoresis
chamber!
What is electrophoresis?
Electrophoresis refers to a
separation technique in which an
electrical field causes charged
molecules to move through a matrix
(usually a gel). Electrophoresis is
routinely used to separate DNA, protein
and other polymeric molecules.
Applications include forensics and
biotechnology research.
Electrophoresis of DNA
Electrophoresis Equipment
The power supply,
black and red cords
leading from the power
supply are attached to
the tray in which the gel
is run, located on the
white benchtop.
Molecular Biology Cyberlab:
http://www.life.uiuc.edu/molbio/geldigest/equipment.html
Gel Casting
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The tray is the actual mold
which provides a shape for
the gel as it polymerizes.
A comb is placed into slots
in the tray, with the
"teeth" down, when the
agarose is still hot. The
agarose polymerizes (after
about 15 minutes) with
small "wells" in it into
which samples are added.
The comb is gently lifted
up out of the gel after
running buffer has been
poured over the gel.
Adding the samples
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5-10 µl is all that is
typically loaded into
each well
Power Supply +
-
Cathode –
Anode +
(Red)
Electrophoresis Chamber
(Black)
500bp DNA ladder
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Contains 16 bands
in 500bp increments;
the smallest one is
500 bp
Used to estimate
DNA fragment sizes
using a 0.8% - 1.0%
agarose gel.
The concentration of an agarose gel allows for
the separation of different sizes of DNA
molecules
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0.5%
1.0%
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100Kb
20Kb
20Kb
(50bp)
.5Kb (500bp) .05Kb
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2.0%
4Kb (4000bp)
Voltage
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The higher the voltage, the faster the
rate of migration. However,
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Accompanying heat may melt the gel.
(Don’t use more than 125 volts).
Imperfections in the gel distort the bands
and produce ambiguous results (slants and
smiles).
Electrophoresis
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Enzyme will cut 160
base pair normal
protein into two 80
bp fragments; will
not cut mutated
protein.
Measure against a
100 bp ladder
What are the
genotypes of the
father, mother, and
their newborn child?
Gel stained with ethidium bromide
and visualized with UV light
Electrophoresis
Ladder
Diagram
Hemoglobin: A Molecule to
Breathe By
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In lungs:
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In tissues:
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Hb + 4 O2 ----------------> Hb.O8
Hb.O8 -----------------> Hb + 4 O2
In the tissues, some Hb picks up CO2
(~25% of total) and transports it back
to the lungs where it is released.
Basic Hemoglobin Structure
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2 alpha globin subunits
2 beta globin subunits
Each globin subunit consists of 8 alpha
helices folded together into an identical
shape.
Each globin subunit contains an
identical heme group.
Model of the beta globin chain
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The heme group (in red) is held in place by interactions with
histidine side chains (shown in green). The iron atom is located
in the middle of the heme molecule.
Protoporphyrin and Heme
Hemoglobin’s affinity for
Oxygen
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Fe 2+ (in middle of the heme group)
lies slightly below the plane of the
ring.
When bound to O2,, Fe lies in the
plane of the heme group.
Heme absorbs green and yellow
wavelengths; reflects orange and red.
The Genes that produce Adult
Human Hemoglobin
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2 α globin: gene on chromosome 16
2 β globin: gene on chromosome 11
Alpha Globin (HbA) Gene Locus
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Chromosome 16
2 ζ genes expressed only first few weeks of development
2 α genes expressed thereafter
5’----ζ2--ζ1--α1--α2--α1----3’
Beta Hemoglobin
(HbB)
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Gene loci: 11p 15.5.
3 exons (coding regions)
scattered over 1600 base pairs
Yields a 626-bp mRNA
transcript
Translated into a 147 amino
acid polypeptide
Beta Globin (HbB) Locus: multiple genes
arranged sequentially from 5’ to 3’
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Epsilon ε – expressed during first trimester
Gamma γ – “ during fetal development
Delta δ - “ in small quantities
Beta β – most abundant
5’--- ε---Gγ--Aγ---β1—δ---β---3’
Human hemoglobin
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Embryonic: 2ζ, 2ε; 2α, 2ε
Fetal (HbF): 2α, 2γ
Adult (HbA2): 2α, 2δ
Adult (HbA): 2α, 2β
HbF has a much higher affinity for oxygen
than HbA. A significant amount of HbF
persists for ~8 months after birth.
Bioinformatics…
The Evolution of Hemoglobin is a
story of…
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Duplications
Mutations
Transpositions
Over billions of years through plants and
all animals (see color page)
Evolution of Hemoglobin
http://www.people.virginia.edu/~rjh9u/slidlist.html
Anemia
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Any significant decrease in amount of
functional Hb
Due to a number of causes
All forms have serious physiological
effects
Sickle Cell Anemia:
A Series of Firsts
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1st known molecular disease. Thousands of such
diseases (most rare), including over 150 mutants of
hemoglobin alone, are now known.
1st disease by which electrophoretic analysis was
applied (by Linus Pauling and Harvey Itano).
Microscopic observations showed that individuals with
sickle cell trait had about half normal and half sickle
cell hemoglobin
1st verified case of a genetic disease that could be
localized to a defect in the structure of a specific
protein molecule.
HbA
HbS
Sickled Red Blood Cells
contain Deoxy HbS
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HbS polymerizes when deoxygenated
More rigid than HbA
Stickier than HbA: adheres to walls of blood
vessels
Clogs the arterioles and capillaries preventing
the blood from delivering oxygen and
nutrients, and removing carbon dioxide and
wastes from the tissues.
Deoxy HbS
DeoxyHbS
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DeoxyHbS molecules lock
together, line up into long
fibers inside the RBC and
become rigid, precipitate
out of solution and cause
the RBC to collapse.
End on view (Stetson, J. Exp.
med. 123:341-346, 1966.)
Longitudinal view of deoxyHbS [From G. Rykes, R.H.
Crepeau, and S.J. Edelstein. Nature 72(1978):509.]
Sickle Cell Anemia
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Genotype: HbS HbS
Short-Term: Frequently out of breath and easily tired
Crisis: acute musculo-skeletal pain caused by loss of
oxygen in tissues
Long-Term:
Hand-foot syndrome: dactylitis damages small bones of
hands and feet during first 2 years of life.
Tissue death in top of femoral bone often crippling
Other complications include stroke, organ damage or
failure, leg ulcers.
Shortened life span
Sickle Cell Trait
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Heterozygous condition: HbA and HbS
Individual is asymptomatic most of the
time
Crises occur when the oxygen
saturation falls below 40% or when
barometric pressure falls to 50 mm of
mercury (1/3 normal level)
Malaria: A 2-host disease
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The sexual stage develops in the mosquito.
The asexual stage (rapid cell division) of the
protozoan, Plasmodium vivax, occurs within
the red blood cells of humans. The RBCs
burst accompanied by a very high fever;
followed by lower temperature and the
sensation of "chills”.
An Adaptive Response:
Resistance to Malaria
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In HbA HbS individuals, half their hemoglobin
will sickle when the oxygen tension becomes
very low. These sickled cells are removed
from the body by the spleen, along with the
merozoites inside of them. Thus,
heterozygotes remove the infected cells from
their body before the protozoans can produce
a large infectious population.
Balanced polymorphism
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Balanced polymorphism: When the
heterozygote in any population is selectively
favored over either homozygote.
In certain parts of Africa today, the
frequency of HbS is very high (10-20%).
HbS HbS may also have an advantage
against malaria, but they have all the other
problems associated with sickle cell disease,
and hence are severely selected against and
seldom reproduce.
Malaria/Sickle Cell Geographic
Correlation
Other Hemoglobin Diseases that
also offer immunity to malaria
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Thalessemia
Ovalocytosis
Favism (G6PD deficiency)
Why is Electrophoresis a Tool?
HbA and HbS have different charges!
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Glutamic acid (in HbA) is acidic
Valine (in HbS) is neutral
Therefore, HbS has 2 fewer negative charges
that HbA – which changes the pH, pI, tertiary
structure, quaternary structure, and oxygen
affinity (function) of hemoglobin. Polymerizes
when deoxygenated.
Glycine, the smallest amino acid
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Main Chain
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Amine group: NH2
Alpha carbon
Carboxylic acid group: COOH
Side group: H –makes it
aliphatic (linear)
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In others, makes it aromatic
(ring), acidic, basic,
hydroxylic, sulphur containing, or amidic
(containing amide group)
Primary Structure of Proteins:
A Review
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Acidic amino acids
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Basic amino acids
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Aspartic Acid
Glutamic Acid
Arginine
Lysine
Histidine
Neutral amino acids – all the rest!
If # of + and – ions are equal: NH3+ = COODepending on the functional groups, the side chains are
aliphatic, aromatic, acidic, basic, hydroxylic, sulphur containing, or amidic (containing amide group).
Acids and Bases
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An acid is a proton donor
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A base is a proton acceptor
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COOH
NH3 +
COONH2
The ratio of acid to base is dependent
upon the pH and pKa.
pH – pKa = log[unprotonated]
[protonated]
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At pH 3, pKa = 0, the ratio of COO- /
COOH is 1/1
At pH 9, pKa = 0, the ratio of NH2 /NH3
+ is 1/1
Isoelectric Point
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The pH at which an amino acid or protein
bears no net charge and, therefore, does not
migrate in an electric field.
pI of neutral amino acids is around pH 6
pI of acidic amino acids is close to pH 3
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Aspartic Acid
Glutamic Acid
pI of basic amino acids is close to pH 9
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Arginine: 10.8
Lysine: 9.7
What happens to an amino acid
in a strong acid solution?
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In an acid solution (excess H+)
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The amine groups become “protonated”
(positively charged)
NH2 → NH3 +
The carboxyl groups are “unprotonated”
(not ionized)
Which way will they migrate during
electrophoresis?
What happens to an amino acid
in a strong alkaline solution?
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In an alkaline (basic) solution (excess
OH-)
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The carboxyls are negatively charged
COOH → COO- + H +
H+ + OH- → H2O
The amino groups are not ionized.
Which way will they migrate during
electrophoresis?
Importance of the Buffer
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The pH of the buffer we will use is 8.6.
Most proteins are negatively charged at 8.6.
Which way will they travel during
electrophoresis?
How fast will they travel?
The rate of migration depends upon its net
charge; the higher the charge the faster the
protein will travel.
Important Links
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http://globin.cse.psu.edu/
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http://www.accessexcellence.org/AE/AEPC/WWC/1993/hemoglo
bin.php
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National Heart, Lung and Blood Institute, National Institute of
Health. Sickle cell anemia: Who is at risk? Available at
www.nhlbi.nih.gov/health/dci/Diseases/Sca/SCA_WhoIsAtRisk.ht
ml Accessed November 3, 2006.
Ashley-Koch, A., et al. Centers for Disease Control and
Prevention. Sickle Hemoglobin (HbS) Allele and Sickle Cell
Disease. American Journal of Genetics. May 1, 2000. Accessed
March 26, 2007.