Mystery of the Crooked Cell:
Investigate Sickle Cell Anemia
Using Gel Electrophoresis
Module developed at Boston
University School of Medicine
Presented by Dr. Dan Murray
Outline
Sickle Cell Anemia
Central Dogma of Biology
Genetic Code
Hemoglobin
Electrophoresis
Sickle Cell Anemia
Sickle Cell Anemia
Genetic Disease
 Heterozygous individuals – carriers
 Homozygous individuals – diseased
Hemoglobin
 Found in red blood cells
 Carries oxygen to tissues
SCA Results from Defective Hemoglobin
 Hemoglobins stick together
 Red blood cells damaged
Complications from low oxygen supply to tissues
 Pain, organ damage, strokes, increased infections, etc.
Incidence highest among Africans and Indians
 Heterozygotes protected from Malaria
Central Dogma
of Biology
Central Dogma of Biology
Transcription:
Conversion of
information from
DNA to mRNA
Translation:
Conversion of
information from
RNA to protein
The Genetic Code
The Genetic Code
start
The Genetic Code
• Protein chains always begin
with Met
• 53 orientation
corresponds to
N-term  C-term orientation
5’
mRNA sequence
3’
AUG AAC AAU GCG CCG GAG GAA GCG GAG
Met---Asn---Asn---Ala---Pro---Glu---Glu---Ala---Glu
Met---Asn---Asn---Ala
Met---Asn
Met---Asn---Asn
Met
N-terminus
Protein sequence
C-terminus
Hemoglobin
Hemoglobin
Multi-subunit protein (tetramer)

2  and 2  subunits
Heme
One per subunit
 Has an iron atom
 Carries O2

In red blood cells
Sickle Cell Hemoglobin
Normal mRNA
Normal protein
GUG CAC CUG ACU CCU GAG GAG AAG
val his leu thr pro glu glu lys
1
2
3
4
5
6
7
8
Mutation
(in DNA)
Mutant mRNA
Mutant protein
GUG CAC CUG ACU CCU GUG GAG AAG
val his leu thr pro val glu lys
1
2
3
4
5
6
7
8
Glutamate (glu), a negatively charged amino acid,
is replaced by valine (val), which has no charge.
Sickle Cell Hemoglobin
Significant change
in structure caused
by the single mutation
A Possible Cure for
Sickle Cell Anemia?
During fetal development, a different
gene (gamma) produces hemoglobin
Expression of gamma gene stops
naturally during development
Research efforts focused on stopping
silencing of gamma gene
Would provide sickle cell patients with
good hemoglobin
Electrophoresis
Gel Electrophoresis
Method for separating molecules (DNA,
proteins, etc.) on the basis of physical or
chemical properties such as:
(1) size
(2) shape
(3) electrical charge
Electrophoresis of DNA
Gels are made of agarose or polyacrylamide
DNA samples loaded, voltage applied
Negatively charged DNA migrates toward “+” electrode
Smaller DNA fragments migrate faster
Electrophoresis of
Proteins
More complex than DNA electrophoresis
Different proteins have different charges
 Proteins vary widely in shape

Polyacrylamide is usually the gel
medium
Protein Electrophoresis
Non-Denaturing conditions
Non-denaturing (native): no pretreatment of
proteins before electrophoresis



Proteins retain normal shape
Proteins retain normal charge
Proteins separated on basis of charge, size, and shape
Name
Charge
Mass
Protein Q
+2
30kD
Protein R
4
42kD
Shape
Non-Denaturing Electrophoresis of
Normal and Mutant Hemoglobin
Charge, Size, Shape
Q. Which of the above properties will be different for
normal Hemoglobin (HgA) and mutant
Hemoglobin (HgS)?
A. Charge: Yes, HgA has one “–” more than HgS.
Size:
No, HgA and HgS are the same size.
Shape: Yes, the shapes are different.
Migration Rates of Normal and
Mutant Hemoglobin
Which Hg migrates faster during electrophoresis?
Normal Mutant
(HgA) (HgS)
Charge
Size
Shape
Reason

HgA has one more “”
than HgS
NA
Amino acids Val and
Glu about same size

NA
HgA more compact
than HgS
Protein Structure
Protein Structure
1 = Primary Structure
2 = Secondary Structure
3 = Tertiary Structure
4 = Quaternary Structure
Primary Structure
Definition - Sequence of amino acids in a
protein
Example – Primary structure of the enzyme
lysozyme:
1
2
3
4
5
126 127 128 129
Lys-Val-Phe-Gly-Arg...Gly-Cys-Arg-Leu
Note: By convention, amino acid sequences are
written starting with the amino terminus.
Secondary Structure
Definition – Regular patterns of relatively
small segments of a protein held together
mainly by H-bonds
Examples:
-helix
-structure
http://www.ultranet.com/~jkimball/BiologyPages/S/SecondaryStructure.html
Tertiary Structure
Definition – Overall 3-D shape of a protein.
Two basic types are globular and fibrous.
Examples:
Globular (Pepsin)
Fibrous
(Collagen)
http://www.ultranet.com/~jkimball/BiologyPhttp://dwb.unl.edu/Teacher/NSF/C10/C10Links/
main.chem.ohiou.edu/~wathen/chem302/protein.htmlages/S/SecondaryStructure.html
Quaternary Structure
Definition – Overall 3-D shape of a multi-subunit
protein
Example:
Rabbit muscle
glycogen
phosphorylase
All Levels of Structure
http://sosnick.uchicago.edu/precpquastru.html
Protein Electrophoresis
Denaturing conditions
Proteins treated with SDS (anionic detergent) before
electrophoresis (SDS-PAGE)
 SDS molecules bind to the Protein
 Proteins lose normal shape
 Proteins all have same charge/mass ratio
 Proteins are separated on basis of size only
Charge Mass
+3
30kD
4
42kD
Charge Mass
SDS treatment
300 30kD
420 42kD