ELECTROPHORESIS

Introduction
Fundamentals of Protein Electrophoresis

 Movement of Charged Particles

- Any charge particle (positive or negative) in an electrical field will migrate to one pole or the other
- Positive charge particle (CATION), will move toward the negative pole (CATHODE) 
- Negative charge particle (ANION), will move toward the positive pole (ANODE)  
- The migration of the charged particles depends on different factors:   
1. Amount of charge on the molecule  
2. Size and shape of the molecule  
3. Strength of electric field   
4. Support medium used in the electrophoresis procedure

 Protein Structure and Electrophoretic Mobility

- Proteins are composed of amino acids and certain amino acids have side chains which may
possess an electric charge at a given pH  
- Each protein has a specific net charge on the molecule depending on the number of these charged
groups present 
- Electric charge is not the only influence on protein migration, although it is a major contributor  
- Molecular shape also plays a role on how far a given protein moves
- With molecular shape, one cannot really make general comments because various techniques are
affected in different ways by the protein shape (whether globular, fibrous or something in between)

Components of Electrophoresis System

Subtopics: There are three basic components to any electrophoresis system
1. Support medium
2. Electrophoresis chamber

- The sample on the support medium is then placed in the electrophoresis chamber.
- The ends of the medium are placed in buffer in separate buffer partitions; the support material then
serves as a bridge between the two areas of buffer.
- Wiring of the chamber allows passage of electric current into one buffer chamber, where the electric
flow then goes across the support material and into the second buffer chamber.

3. Power Supply

- The power supply converts alternating line current into direct current for the operation of the system.
- Most systems have safety mechanisms so that when a component of the apparatus is removed, the
current is cut off, reducing the risk of electric shock to the operator. 
- This piece of equipment also often plays a step-down role
- Voltages across the apparatus are frequently lower than those supplied by the line current.
- Most power supplies provide constant voltage regulation so that the electric field remains very
stable throughout the analysis.
- Today, most electrophoresis systems are marketed as a whole unit, with the chamber and power
supply both being specifically designed for the support medium.

Detection of Proteins after Separation

The main component to any effective electrophoretic technique is its ability to detect the separated proteins
on the support medium.

COLOR DYES
 oThe use of nonspecific protein stains is the traditional method of detecting proteins. Ex.
Amido black and S ponceau
o These dyes essentially attach with all proteins therefore its color is observed wherever the
proteins are.
ENZYME REACTIONS
o If enzymes are being studied, a reaction associated with the enzyme’s function can be used
to highlight sites of interest on the support medium.
o With this method, the location and identify specific proteins without the “noise” from other
materials
IMMUNOFIXATION
o Immunofixation is a process of using specific antibodies to locate proteins that are not
enzymes which is followed by staining to visualize the protein bands.
o Immunoelectrophoretic obtains patterns that are complex and difficult. With the development
of high-resolution protein electrophoresis, immunofixation became possible as an alternative.
o It is simple, yet powerful, for protein identification in body fluids.
o High-resolution electrophoresis on agarose is used to separate protein fractions as the first
stage in the immunofixation process. This allows separation of 12-25 distinct bands.
SILVER STAINING
o This technique is very sensitive for the identification of extremely low levels of proteins.
o This is particularly useful for urine and cerebrospinal fluid (CSF) protein electrophoresis.

Table: Identification of Protein Fractions after Electrophoresis

Quantitation of Protein Fractions by Densitometry

 Various methods have been used over the years to determine the concentration of a certain protein
(or proteins) following electrophoresis and staining. A straightforward visual inspection is sufficient
for some tasks. There is no need for additional information beyond noting the presence or absence
of specific fractions. Rarely is this kind of report helpful in clinical settings. Although it takes a lot of
time, cutting the electrophoresis strip and eluting each fraction into solution for additional chemical
analysis produces results that are reasonably precise. The method of choice in the clinical
laboratory is densitometry, the measurement of the density of light passing through the fractions.
  Most densitometric systems are based on colorimetry, protein fractions are stained with a material
which produces visible bands of colors on the support medium, the strip is placed in a holder and is
slowly moved through a beam of light. Most instance filters are used to permit only light of a certain
wavelength range to strike the strip. As the light interacts with the dye bound to the specific protein
fraction, a certain amount of light is absorbed, depending on the concentration of the protein and
amount of dye bound to it. Absorption of light is roughly proportional to concentration of dye
(concentration of protein fraction)
 The readout from the system is a trace which relates the amount of light absorption to the location
of protein fractions. The data can be further analyzed electronically to provide a percentage
distribution of each fraction on the electrophoresis strip.
Electrophoresis Support Media and Techniques
1. Paper 

 Earliest support medium used for clinical studies (1950)

 ZONE ELECTROPHORESIS TECHNIQUE – gave the first clear picture of serum proteins in health
and disease

Disadvantages 

 Fragile and easily damaged

 Quite porous: significant variability in the degree of separation and reproducibility of results
 Staining of protein fractions was quite variable owing to inconsistencies in the composition of
paper
 Due to the mechanical properties of paper, albumin will be underestimated and other
fractions are overestimated 

2. Starch Gel 
  Developed to overcome the inadequacies of paper
 STARCH GEL ELECTROPHORESIS is still employed in some research applications but never
found a role in the clinical laboratory
 
Advantages 
 
 More uniformity in support medium from lot to lot
 Larger samples could be employed
 
Disadvantages

 Fragile
 Inability to permanently store results
 And the need to prepare the gels in-house

3. Cellulose Acetate
 A more satisfactory support medium was developed in England in 1957, cellulose acetate
electrophoresis 
 This method then becomes the standard method for protein electrophoresis not until it was
replaced by a superior technique in 1970’s

Advantages

 The strip consists of a clear plastic backing with a coating of cellulose acetate particles 
 Size and structure of the particles are controlled 
 Protein is applied as a narrow band onto the strip
 Strip can be cleared of Cellulose acetate, leaving the developed protein bands on
transparent background
 Permanent record of each electrophoresis can be maintained 
 Greater resolution  
 Faster separation 
 Temperature and others factors are closely controlled 
 Uniform particles, bands are sharper and better separated
 Albumin does not absorb to Cellulose Acetate to any extent

Disadvantages

 When dried, the material becomes extremely brittle making it hard to work with and store
 Some chemicals used to prepare the Cellulose acetate strips inhibit developing reactions,
particularly those for the visualization of enzymes 
 If fluorometric development techniques is employed, background fluorescence of the
Cellulose acetate must be compensated
4. Agarose

 The use of agar as an electrophoresis support medium was at the year 1920s
 It was in a 9 foot long, in an agar-filled tubes where separation of isotopes were attempted and it
took several days for the procedure
 With the popularity of agar used a derivative of which, gained popularity as an electrophoresis
support medium
 AGAR - a mixture of polysaccharides derived from seaweed, agarose is predominant of that mixture

Major advantage is its electric neutrality

 There is minimal electric charge on the agarose polymer itself, so little or no interaction with proteins
is expected.
 If agarose fraction will be purified this will result in greater uniformity of materials and better
reproducibility of electrophoretic patterns
 The procedure is much the same as Cellulose Acetate
 Samples for serum protein fractionation require an application of volume of less than 1μL
 For urine and CSF samples require preconcentration and possibly a larger sample size
  If isoenzyme fractionation is desired, samples greater than 1L are frequently required
 Uses buffer or dilute barbital buffer at pH 8.6, where all the charge on most protein molecules is
negative 
 The protein stain of choice for agarose is Amino black
 Certain modifications are now made like the buffer and the apparatus which permits detection of 12
to 15 different protein fractions.
 The High-Resolution protein electrophoresis is increasingly becoming the accepted approach to
routine protein fractionation in CC

Polyacrylamide Gel Electrophoresis (PAGE)

 The size of the molecule had a minor impact on protein fractionation using paper and cellulose
acetate as support media, but the charge on the molecule was the primary determinant of
electrophoretic mobility. Protein size is unlikely to have any impact on protein separation on
agarose.
 The greater the molecular charge, the faster the protein moves through a gel as it is propelled by an
electric current. Smaller molecules are not as severely slowed down by the gel's sieving effect. The
end result is a separation of protein molecules that is more precise.
 Hemoglobin, lipoproteins, and certain isoenzymes were once commonly separated using PAGE. For
routine work, agarose has mostly replaced this method due to the intricacy of the process
(particularly in the gel preparation steps). However, PAGE has continued to demonstrate its value
as a method for protein separation in a variety of specific applications.

Procedure:

- As chon in the sample migrate through this polymer, driven by the electric current, some of the chon
are held up more than others by the polymer matrix. 
- Large molecules find it more difficult to move through this system, but smaller molecules are not
retarded as much by the sieving effect of the gel.
 Therefore; for a given total molecular charge, the smaller molecules moves faster than the larger
one through the gel
 Combination effect of: 

- The greater the molecular charge, the faster the chon moves. Therefore: the larger the molecular
size the slower its movement through the gel. The result would be a more detailed separation of
chon molecules and a more complex pattern resulting from the separation.

Isoelectric Focusing
Isoelectric point 
          Is the pH where the net charge in the molecule is zero
 
 Other method of separation was based on the aspect of the net charge of the molecule; this method
exploits a different parameter associated with the chon charge, the Isoelectric point.
 The net charge on the chon is negative at PH values greater than the isoelectric point, positive at
lower PH values, and zero at the isoelectric point for the molecule.
 Isoelectric point does not cause the chon to become electrically neutral, but the different local
charge at different points on the chon can close each other, providing a net charge of zero for the
whole molecule.

Ampholyte
          A structure with 300 to 600 molecular weights. To give the ampholyte the desired charge at the
specified pH, different numbers of carboxyl and amino side chains are integrated into the molecule. 
 
 When a gel is prepared which contains carrier ampholytes, the pH in one area of the gel is not the
same as it is in another region. It is impossible to generate pH gradients as small as 0.001ph unit
within a gel with the proper selection of ampholytes.

Procedure in Selecting Ampholytes:

 To perform isoelectric focusing, protein and ampholytes are mixed together and applied to the gel.
  The ampholytes distribute themselves across the gel according to their pH values.
  As the protein moves across the gradient, it slows down as it enters a region close to its isoelectric
point. 
 When the molecule moves into a region where its net charge becomes zero, migration stops. 
 If it diffuses into another area, the net charge on the molecule changes and the protein migrates
back to its isoelectric point under the influence of the electric field. 
 The resulting pattern is a series of very fine bands of protein, each one separated from the others
on the basis of its unique isoelectric point

Major Use of Isoelectric Focusing:

1. Major uses appear to be in the study of immunoglobulins, either in the characterization of the
normal molecules or in the fractionation of these CHON in clinical situation.
2.  Examination of various abnormal CHON found in CSF in patients w/ multiple sclerosis
3. Study of isoenzyme distribution, example would be ALP (Alkaline Phosphatase) isoenzyme in
various body fluids 
 characterization of the normal molecules
 fractionation of proteins in clinical situations

Isoelectric focusing has been used to examine CSF in patients with multiple sclerosis, as well as for the
study of isoenzyme distribution. This technique has proved to be of great value in examining the various
abnormal proteins found in CSF from patients with multiple sclerosis.

Two- Dimensional Protein Fractionation

 Five main fractions of interest can be identified through routine protein separation on agarose. At
the very least, high-resolution electrophoresis doubles that capacity. 
 Even more protein fractions are shown by the increase in separating power if we move to
polyacrylamide gel or isoelectric focusing.
  However, a variety of proteins can be found in various body fluid. The possibility to examine the
majority of these thousands of proteins in a single analysis is made possible by the combination of
two different methodologies, given that a single technique cannot detect all these various entities.

Two- dimensional Protein Electrophoresis

 Isoelectric focusing and polyacrylamide gel electrophoresis are two methods that have been
combined and modified to create two-dimensional protein electrophoresis.
  In a tube gel, proteins are first separated according to their isoelectric points. After that, the
fractionation is moved to a slab for further polyacrylamide gel processing. 
 Silver staining is typically used to identify patterns. A theoretical resolution of over 10,000 proteins is
possible with this analytical approach.
 Sophisticated computer-driven scanners have been developed to examine the patterns, store the
data, and do pattern comparison to quantitate materials and to detect the presence or absence of
specific proteins. ]
 Although two-dimensional electrophoresis is currently a research tool, it may be of great value in
studying some clinical situations.

Problems Associated with Protein Detection and Measurement

1. Buffer Concentration and Ionic Strength
- The motility of proteins depends greatly on buffer concentration and the total charge composition of
the system
 Example: If there is a decrease in buffer concentration, produces an increase in mobility of
protein fractions, with a broadening of each band. 
- The ionic strength is related to buffer concentration and to the types and amounts of ionic charges
present.
 The higher the ionic strength, the slower the movement of protein fractions
- Not only buffer concentration, but also buffer type. As there are some buffers that may have the
same molar concentration but very different ionic strengths owing to the specific composition of
each buffer.
 2. Electroendosmosis

- It is a phenomenon relating to induced charges in the medium.

- To achieve optimum separation of protein fractions, we must minimize electroendosmosis.
- The interaction of charged protein molecules with the charged groups on the medium alters the
mobility of the protein to some extent.
- This change in migration rate may be reflected in a displacement of the band. It either moves ahead
of or behind its usual location
o More likely we see a band distortion (a wavy fraction) or spreading
- The charged particles on the solid support matrix also affects the buffer ionic strength.
-  As the buffer ions move, the ionic strength changes, resulting in altered mobility of protein fractions
and distortions in the resulting bands.

3. Heat and Voltage Effects

- More voltage in the electrophoretic system, we move the proteins (CHON) more rapidly
- Increase current meaning increase in temperature
- Several problems arise as temperature begin to rise, protein (CHON) mare labile molecules, easily
denatured by higher temperature (50℃ or higher)
- Mobility of proteins (CHON) fractions are enhanced resulting to a band in a different location than
the one set at lower temperature
- Evaporation of solvent increases at higher temperature, causing an increase in ionic strength of the
buffer
- This increase can be noticed along the edges of the electrophoretic strip, where distortion and
trailing of the band are likely to occur
- In high resolution, a cooling system is incorporated to resolute the temperature

4. Staining and Scanning Problems

- There are varying concentrations of the different protein (CHON) and different structures resulting in
different dye-binding capabilities
- Some proteins (CHON) require specific developing agents and other can be detected on the strip or
tube without the need for visualizing reagent
- Conditions for staining and observing protein (CHON) bands must be carefully selected to achieve
the desired purpose
- Concentration of the stain and reaction time needs to be selected to allow development of all bands
present
- A protein (CHON) in low concentration may react sufficiently in a given time, another protein
(CHON) present at a much higher level may have inadequate 
- The background contributed by Densitometric Scanning
- Scratches, spots or local discolorations falsely after the levels of certain protein (CHON)
- All methods need to be examined carefully to ensure that artifacts are not introduced leading to
erroneous result (values) for protein (CHON) quantitation 

Serum Protein Electrophoresis

1. Patterns obtained with Various Techniques

- Some similarities and differences of protein fractions can be seen, as well as identified, using
various electrophoretic techniques.
- In the standard method for serum protein electrophoresis (SPE), serum samples are applied close
to the cathode end of a support medium that is saturated with an alkaline buffer with a pH of 8.6.
The support medium is connected to two electrodes and a current is passed through the medium to
separate the proteins. All major serum proteins carry a net negative charge at pH 8.6 and migrate
toward the anode. Using standard SPE methods, serum protein appears in 5 bands (order of
migration in SPE):
 1. Albumin – travels farthest to the anode
2. α1-globulin
3. α2-globulin
4. β-globulin
5. γ-globulin

After separation, the protein fractions are fixed by immersing the support medium in an acid solution (to
denature the proteins and immobilize them on the support medium) and then the proteins are stained using
a variety of dyes such as Ponceau S, Amido black, and Coomassie blue. The result would be the
appearance of a band on the support medium which are the proteins. The width of the band of proteins in a
fraction depends on the number of proteins present in that fraction. And homogeneous protein gives a
narrow band.
- No clear and consistent pattern is available on PAGE, isoelectric focusing, and two-dimensional
techniques.
- References to protein fractions are generally based on mobilities and properties defined by the
classic agarose approach.

2. Protein Fractionation on Agarose

- also called the “5-band” approach; protein fractionation on agarose using classical methods yields
five bands.
- Although high resolution produces additional bands, the overall fractionation pattern of albumin
(most rapid migration among 5 bands) still holds.
- Protein fractions/zones:

1. Prealbumin fraction
- contains two proteins which bind thyroxine and alpha2 macroglobulin
- the derivative of a drug that has attached itself tightly to albumin may be present here also
2. Albumin fraction/zone
- largest single contributor to total protein level in body
- occasionally contain a separate prealbumin component which is mostly not visible
- Consists essentially of one protein – albumin
- Variations are due to genetic effects on protein structure or binding to albumin by other molecules
which may result in either two bands in the albumin region or a broader band than usual; a
phenomenon known as Bisalbuminemia
3. Alpha1 -globulin zone
- major protein is alpha1 antitrypsin which is responsible for inhibiting proteolytic enzyme trypsin, and
other related enzyme, activity
- another protein in this zone is lipoprotein which usually migrates closer to albumin
- other minor proteins are often seen in this zone or in between this component and the next (alpha 2 -
globulins)
4. Alpha2 -globulin zone
- role is largely unknown; evidence suggest that it may bind trypsin and insulin
- contains physiologically significant proteins such as:
a. Ceruloplasmin – copper transport
b. Lipoproteins
c. Haptoglobin – responsible for binding free hemoglobin
d. Alpha2 macroglobulin – protease inhibitor
 
5. Beta globulins fraction
- a heterogeneous mixture of proteins
- important components in this fraction include:
a. Lipoprotein
b. Transferrin – responsible for binding and transport of iron
c. Hemopexin – responsible for transport of free heme
d. Complement system – key part of immune response; functions in: (1) the activation of
inflammation; (2) the opsonization (labeling) of pathogens and cells for clearance/destruction;
(3) the direct killing of target cells/microbes by lysis.
6. Gamma globulins fraction
 - most immunoglobulin are found here; some may be located in the beta globulin fraction
- change in concentration of gamma globulin is often the first indicator of issues with the
immunoglobulins found here such as:
a. IgG – most common antibody; enhance the phagocytosis of pathogens, neutralize bacterial or
viral toxins, and trigger the activation of the complement system
b. IgA – present in blood, lymph and other body secretions such as saliva, tears and milk; protects
against bacterial growth and colonization. It is less stable than IgG and can be found in lower
quantity
c. IgM - first-line defense against a broad range of infections
d. IgD – less than 0.5% of serum antibodies; found in lesser amounts in lymphatic fluid and blood;
function still unknown
e. IgE – most effective antibody against parasitic infections; less than 0.01% or serum antibodies

Table: Significant Proteins in Electrophoretic Fractions

Clinical Applications of Serum Protein Electrophoresis

General Principles  
 Protein electrophoresis pattern can provide quick and useful information regarding altered levels of
chon groups within the circulation  
 Some disorders have characteristic pattern which are readily recognized  
 Most situation, the role of electrophoresis pattern in providing specific clinical information is limited  
 Many patterns are somewhat nonspecific; more than one disease state produces the same
electrophoretic distribution of chon  
 Although a qualitative change in the level of a specific chon may be observed, the electrophoretic
data are not accurate enough to deal with the exact amount of change, but can provide some
measure of specificity  
 Interpretation of chon electrophoresis patterns in the absence of detailed clinical information about
the patient is fraught with hazards and should be avoided.
 Because of the complexity of data and possible interpretations only a brief overview of the diagram
of diagnostic possibilities will be presented here.

Table: Serum Protein Electrophoresis Changes in Common Disorders

1. Inflammation
 Number of biochemical changes occurs during inflammation that is caused by fever and infection

Acute Phase
 Body responds to the stress by increasing the concentrations of several proteins involved in dealing
with infectious agents.

Chronic Phase
 Arises from a variety of sources including chronic infection, allergies, collagen disease, and related
disorders, malignancies, and autoimmune problems.

 Serum Protein Electrophoresis

- An easy inexpensive method of separating proteins based on their net charge, size, and shape. 
- The 2 major types of protein present in the serum are albumin and the globulin proteins. 
 Albumin is the major protein component of serum and represents the largest peak
that lies closest to the positive electrode.
 Globulins make up a much smaller fraction of the total serum protein but represent
the primary focus of interpretation of serum protein electrophoresis

Figure 6-8. Serum Protein Electrophoresis - in acute inflammation.

Figure 6-9. Serum Protein Hypothesis- in chronic inflammation.

2. Hepatic Disorder

 In liver disease of whatever cause, a major result of the hepatic damage is a decrease in protein
synthesis.
 Many serum proteins, commonly albumin, are manufactured by the liver.
 If complications were to arise from this organ low levels of albumin, lipoproteins, etc are expected to
occur.
 Increase in the number of gamma globulin indicates an infection in the body.
 -Patients with hepatitis manifest elevation of IgG,IgA, IgM.
-In cirrhosis the characteristic pattern is a single broad in place of the distinct separation between
the beta- and gamma globulin fractions.
 The bridging effect is due to the enhanced production of the IgA.

3. Renal Protein Loss

4. Lipoproteins
5. Immunoglobin Abnormalities

 Serum protein electrophoresis cannot be used to quantitate the levels of the different types of
immunoglobulins. 
 We can roughly determine the overall immunoglobulin situation by examining the gamma-globulin
fraction alone.
 More specific techniques are required to quantitate any particular immunoglobulin. 
 The major role of electrophoresis in the study of immunoglobulin abnormalities is to shed light to
various abnormal fractions seen in serum or CSF. These atypical proteins are easily observed by
electrophoresis, triggering further specific tests to identify the details of the underlying pathological
state more clearly.

Limitations of Electrophoretic Data

Before we developed the capability of assaying for specific proteins in serum or other body fluids, the major
test which allowed any sort of assessment of detailed protein status was electrophoresis. 
 
 We could gain useful information about classes of proteins, but in most cases were not able to
quantitate any one protein with accuracy. 
 Protein electrophoresis no longer enjoys the premier status it once did. Electrophoretic patterns are
now used as screening tests, to suggest further evaluation of a fraction or fractions by selective and
specific analyses. 
 There is still a great deal of debate about the relative merits of electrophoresis and specific protein
assays, but more and more evidence (as well as changes in laboratory utilization and
reimbursement patterns) points to the value of determining the quantity of certain proteins in any
given disease state.

Urine Protein Electrophoresis

Urine protein electrophoresis (UPEP) is a test which doctors utilize to determine if there's protein in
a patient’s urine. It can also aid the doctors to find out how much of each type of protein is present.

Normal Studies
 A healthy person is usually characterized by a low level of a total protein and electrophoretic pattern
(on AGAROSE) that is diffuse and nondescript.
 A small amount of albumin and other low molecular weight components may be observed.
 Quite often, the urine sample needs to be concentrated several folds before any distinct bands can
be detected.

Clinical Utility
 The analysis of urine protein with two-dimensional electrophoresis reveals presence of over 250
specific protein fractions.
 Urine protein electrophoresis can point out whether proteinuria is due primarily to albumin loss or to
increased excretion of other proteins.
 Comparison of the urine electrophoresis pattern with a serum pattern from the patient may indicate
something about the pattern of protein loss.

Cerebrospinal Fluid (CSF) Proteinelectrophoresis

Cerebrospinal fluid (CSF) is a test in which immunoglobulin protein subtypes are separated using an
electrical current applied to a CSF sample. The CSF often does not have a lot of protein. An inflammatory
or immunological condition may be indicated by a rise in the levels of these proteins, these include
meningitis and multiple sclerosis (MS).

Normal Studies
 It has a total protein level of up to 45 mg/dL. This low amount indicates that concentration of the
fluid is necessary before clearly discernable fractions can be seen on protein electrophoresis
 The cerebrospinal fluid patterns are very much like serum, with the predominant fraction being
albumin. The major difference between serum and CSF is the presence of a very clear prealbumin
fraction in samples from spinal fluid, amounting to some 4-5% of the total protein.
 CSF protein electrophoresis pattern can be distorted if there is difficulty in obtaining a clear
specimen. These proteins obtained from the blood with the spinal fluid seriously affect the results.

Clinical Utility (CSF)

 The major purpose of performing CSF protein fractionation is to obtain information regarding the
production of abnormal immunoglobulin bands seen in multiple sclerosis. 
 Abnormal electrophoresis patterns in CSF may be seen secondary to changes in serum fractions.

Hemoglobin Electrophoresis and Hemoglobinopathies

Hemoglobin Structure and Function
Hemoglobin

 major constituent of erythrocyte, red blood cells

 is a protein and responsible for the transport of oxygen throughout the circulation (of the body)
 Composed of:
- 4 polypeptide chain
- 2 alpha chains
- 2 beta chains
 The hemoglobin molecule is formed when the four peptide subunits interact with each other.
 The Formation of alpha and beta chains is regulated by separating the genes; each type of chain is
synthesized independently of the other type.
 Chains also contain a porphyrin ring with an iron atom in its center.
 The iron atom is a site of binding for oxygen.

Porphyrin ring
The porphyrin is the framework for the heme molecule, the pigment in hemoglobin and the red
blood cells. It’s also a large ring molecule consisting of 4 pyrroles, which are smaller rings made from 4
carbons and 1 nitrogen.
 Structure of the hemoglobin

 In the lung, the oxygen attaches to the hemoglobin at the porphyrin site.
 Each hemoglobin molecule has the capacity to bind four oxygen molecules, one at each porphyrin.
 As the oxygen attaches to the porphyrin, the three-dimensional shape of the protein changes.
 The more oxygen which attaches, the more the protein conformation is altered. When the oxygen
leaves the hemoglobin molecule, the shape returns to its original form. 

Three Fractions of HGB Found in the Normal RBC:

1. Hemoglobin A
- Adult hemoglobin or hemoglobin A1
- Is the major component which carries the oxygen to the body
- Approximately 95% of the total hemoglobin.
- Composed of two alpha and two beta chains

2. Hemoglobin A2

- is a normal variant of hemoglobin A  

- Consists of two alpha and two delta chains
- it’s found at the low levels in normal human blood
- Comprises up to 3.5% of the total hemoglobin

3. Hemoglobin F

- Fetal hemoglobin 
- HbF is the main oxygen carrier protein in the human fetus
- The final 2% of the total hemoglobin
- Two alpha and two gamma chains
- it is found in high concentrations in the red cells of newborns

A wide variety of genetic disorders

- In nucleic acid coding for protein synthesis, an error alters one of the alpha chains or beta chains
amino acid sequence as a result.
- The hemoglobin molecule is frequently less able to function normally as a result of these alterations.
 - Therefore, in the end, these abnormal processes may cause oxygen transport to be impaired, red
blood cells to break down sooner than normal, and cellular damage to occur.
Hemoglobin Electrophoresis

If the altered amino acid sequence produces a difference in the total charge on the hemoglobin molecule,
the abnormal hemoglobin can be separated from normal components by:

 Electrophoresis.
o The initial step in the process involves removal of hemoglobin from the erythrocyte.
o Blood is collected with an anticoagulant (several are suitable) and centrifuged to separate red
cells from plasma.
o After the supernatant plasma is discarded, the cells are washed once with 0.155 M NaCl and
centrifuged again.
o Distilled water (1:9 ratio of cells to water) is then added to the cellular precipitate to lyse the red
cells.

Centrifugation removes the stroma (red cell wall), leaving the hemolysate ready for electrophoresis.

The protocol for hemoglobin electrophoresis is similar to that for serum protein separations.

Fractionation of hemoglobin takes place at pH 8.6 on either cellulose acetate or agarose. Either S
Ponceau or amido black may be used for staining after the separation is complete.

Since fractionation at pH 8.6 does not always reveal every abnormal fraction, a second separation at pH
6.2 on citrate agar is strongly recommended to obtain further data.

Hemoglobin Electrophoretic Patterns

 Detects a variety of hemoglobinopathies, group of disorders passed down through families
(inherited) in which there is abnormal production or structure of the hemoglobin molecule.

NORMAL HEMOGLOBIN:

 At pH 8.6, adult Hemoglobin (Hb A) migrates the furthest toward the anode (+). Hb F is found
somewhat behind Hb A, but minor adult Hemoglobin (Hb A2) does not migrate very far from the
origin or point of Application, cathode (-).

 Several abnormal hemoglobin fractions (including Hb S) are found between Hb A 2 and Hb F.

However, these hemoglobin fractions: Hb C, Hb D, and Hb E, travel the same distance as Hb S and
cannot be fractionated at this pH.
  At pH 6.2, however, fractionation on citrate agar at pH 6.2 allows separation of hemoglobin fractions
S, C, and D (among others). Although Hb A still moves toward the anode at pH 6.2, both Hb C and
Hb S travel in the opposite direction. Hemoglobin S does not move far from the point of application,
but Hb C moves further toward the cathode. Hemoglobin D comigrates with Hb A in this system, and
Hb F moves the furthest toward the anode.

Sickle Cell Disorder

 It is produced because of the presence of Hb S (an abnormal hemoglobin in which the sixth
amino acid in the beta chain is valine instead of glutamic acid) in the erythrocyte.

What causes the characteristic sickle shape of the cell?

 When oxygen is removed from the Hb S molecule, the shape of the molecule changes which allows
adjacent beta chains to interact and form long, insoluble polymers inside the red cell. Such
polymers precipitate from the solution and distort the shape of the erythrocyte

Two forms of the Disorder Exist

1. Sickle Cell Trait

 The individual having this trait is heterozygous for the disorder: he/she inherits the Hb S gene from
one parent and the normal Hb A gene from the other parent.  
 35% Hb S with the remainder normal Hb A is shown for the hemoglobin electrophoresis pattern for
this situation.

 Individuals with the trait may not manifest any significant medical problems over the course of
their lives.
 Occasionally, however, a patient may experience some problems in a lowoxygen environment. But
this is an infrequent occurrence.

2. Sickle Cell Disease

  This involves the inheritance of the Hb S genes from both the parents, meaning to say, the
individual is homozygous for this disorder.  
 In electrophoresis studies, only Hb S gene is shown with no trace of the Hb A gene.
 Higher-than-normal levels of Hb F are also seen in patients with this disease, frequently.

Thalassemia

 Comprises a series of hemoglobin disorders in which one of the normal subunits is synthesized
only in extreme low concentrations, or is not produced at all, as opposed to sickle-cell
disorders, where an abnormal protein is synthesized.

Overall clinical effect: greatly diminished formation of red cells, with all the complications of severe
anemia.

Thalassemia Cause
Alpha thalassemia Impaired formation of the alpha
chain
Group of beta-thalassemia syndromes Defects in beta-chain synthesis
Formation of Excess production of the
hemoglobin tetramers: unaffected subunit
Hb H (four beta
subunits)
Bart’s Hb (four gamma
subunits)

Screening for thalassemia:

•      Variety of hematology tests, in addition to hemoglobin electrophoresis

•  Hemoglobin F and A2 analyses – crucial since these two fractions are frequently quite elevated in
thalassemia
•      Electrophoresis may show an abnormal hemoglobin (Hb S is seen frequently), suggesting a
mixed disorder.
•  Studies of the individual globin chains provide useful information, but are usually carried out by a
reference laboratory. 

Other Disorders of Hemoglobin Production

 Over 300 different hemoglobin mutations are presently known.

 Amino acid substitution (such as of the sickle-cell disorders) does not result in any deleterious
effect.
 Genetic abnormalities resulting in synthesis of Hb C and Hb D are of clinical significance, since
these disorders do have an adverse effect on the health of the individual.
 Association of the clinical problems in any given hemoglobinopathy:
 o Stability of the hemoglobin molecule
o Amount of hemoglobin available
o Problems with formation of sufficient numbers of erythrocyte 

Summary
The fact that charged particles migrate in an electric field; this becomes an advantage for Protein
Electrophoresis. Appropriate techniques are utilized so that they can be separated as each of the proteins
have net charge. A simple electrophoresis framework consists of;
1. Support/separation medium
2. Buffer or the electrophoresis chamber
3. And a power supply
Proteins can be detected using variety of stains following the protein fractionation. These stains include
amido black, Coomassie blue (a stain that is very useful for urine and CSF protein studies), these also
include silver staining (which is useful for differentiation of proteins in low concentration). The quantitation
of the different protein fractions is done by Densitometric scanning, as defined to be the measurement of
the density of light passing through the fractions.
A wide variety of separation approaches are available. The most commonly employed media are
1. cellulose acetate and
2. agarose
In both methods, proteins separate according to molecular charge, whereas polyacrylamide gel
electrophoresis uses protein fractionation according to size and charge. Isoelectric focusing takes
advantage of the isoelectric point of the proteins. Two-dimensional protein electrophoresis combines
isoelectric focusing with subsequent further fractionation by size exclusion with polyacrylamide gel.
Changes in the concentration of Buffer and ionic strength affect:

- the migration patterns of proteins in different ways

- the heat generated causes pattern distortion and protein fractions are being destructed

Proteins are not stained fairly in the same way with a given staining medium, thus it creates some
difficulties with accurate quantitation or measurement of proteins and the reproducibility result is highly
affected.
The serum protein electrophoresis pattern using agarose or cellulose acetate observed in healthy
individuals consists of a strong albumin peak and fairly well-defined small peaks for the alphas alpha2, and
beta globulins. The normal urine protein electrophoresis pattern consists of albumin with very diffuse and
low levels of the various globulin fractions.
Electrophoresis is a versatile and powerful analytical technique that is capable of separating and
analyzing a diverse range of ionized analytes. The mobility can be a reflection of the characteristics of the
biomolecules or particles of interest or result from their interaction with another molecule. When combined
with other techniques, electrophoresis can create an outstanding and all-around tool that is applicable to
research, routine analysis, and diagnosis.