L04 Plasma Protein PDF

: Fakulti Pergigian
Plasma Proteins
Ts. RD.AIDIFITRINA BINTI KHIROTDIN

B.Biomedical Hons. (Clinical Biochemistry) (UKM) & M.Community Health Sc.(Hos.Management & Health Economics) (UKM)
Professional Technologist MBOT (PT 18050764), LAEP & Technical Assessor, SAMM MITI
Principal College, Students Affairs

& Lecturer (Biochemistry & Multi Disciplinary Hospital Health Management)
of UITM Branch Office Selangor, Sg.Buloh, Selayang and Teluk Intan Campus
Pejabat Rektor, Aras 1, Bangunan Akademik,
Centre of PreClinical Science Studies,

Faculty of Dentistry, Universiti Teknologi MARA Sungai Buloh Campus,
Jalan Hospital, 47000 Sungai Buloh, Selangor, Malaysia
: Fakulti Pergigian
STUDENT LEARNING OUTCOME:

1. Explain the basic of classification of plasma protein
2. Name the major types of plasma protein
3. List the major role of plasma proteins
4. Name the example of disease that use plasma protein as

diagnostic tools
5. Explain the basic principles of Electrophoresis through

experiment
SLO 1. Classes of Plasma Proteins
: Fakulti Pergigian
Albumins - 60%  Plasma contains >300 different proteins

Globulins (α, β, γ) - 35%  Many pathological conditions affect level of
plasma protein
Fibrinogen - 4%
 Mostly synthesized in the liver
Other proteins - 1%  Some are produced in other sites
 A normal adult contains ~70 g/L of pps
12/5/2019 Biochem/copps;dee 3
Site of synthesis of Plasma Proteins : Fakulti Pergigian
▪ Liver - 90% plasma proteins

▪ Plasma cell (B lymphocytes) - γ globulin
▪ Peptide hormones – Endocrine glands
Liver dysfunction - plasma proteins

A:G ratio (normal): 0.8-2.0., Decreased in liver
dysfunction and chronic inflammatory conditions.
: Fakulti Pergigian
Types of Plasma Proteins

• Prealbumin
• Albumin
• α1-Globulins:
• a1-Antitrypsin, α-fetoprotein
• α2-Globulins:
• Ceruloplasmin, haptoglobin
• β-Globulins:
• CRP, transferrin, β2-microglobulin
• γ- Globulins
SLO 2. Major type of Plasma Proteins : Fakulti Pergigian
Functions of pps : Fakulti Pergigian
• Transport (Albumin, prealbumin, globulins)

• Maintain plasma oncotic pressure (Albumin)
• Defense (Immunoglobulins and complement)
• Clotting and fibrinolysis (Thrombin and plasmin)
Functions of plasma proteins
: Fakulti Pergigian
1. Colloid osmotic pressure in blood Colloid osmotic

2. Viscosity of blood
pressure (oncotic
3. Buffer action
4. Clotting and fibrinolysis pressure)
5. Defense function body
6. Transport function 80% of plasma oncotic
7. Plasma proteolytic enzyme system pressure is maintained by
8. Plasma protease inhibitor system albumin.
9. Nutritional reserve
It is the opposing force to
hydrostatic pressure.
: Fakulti Pergigian
Colloid osmotic pressure (oncotic pressure) and filtration of fluid
Edema
Due to disturbance in
hydrostatic and/or
oncotic pressure between
intra-capillary and
interstitial component.
Organ specific : Fakulti Pergigian
• Brain: Cerebral edema

• Lung: Intra-alveolar=pulmonary edema,
intra-pleural=pleural effusion
Clinically
• Peritoneum=ascites ▪Pitting ▪Non pitting
• Severe generalized edema=anasarca
• Leg edema- deep venous thrombosis
Edema▪Pitting ▪Non pitting
12/5/2019 Facial oedema Biochem/copps;dee 10

: Fakulti Pergigian
SLO 4. Name the example of disease that use plasma protein as
diagnostic tools : Fakulti Pergigian
Prealbumin (Transthyretin)
 A transport protein for:
 Thyroid hormones
 Retinol (vitamin A)
• Migrates faster than albumin in electrophoresis
 Separated by immunoelectrophoresis
Lower levels found in:
 liver disease, nephrotic syndrome, acute phase
inflammatory response, malnutrition
 Short half-life (2 days)
Albumin Functions : Fakulti Pergigian
• Most abundant plasma protein • Maintains oncotic pressure:

(~40 g/L) in normal adult • The osmotic pressure exerted by
plasma proteins that pulls water
 Synthesized in the liver as into the circulatory system
preproalbumin and secreted as
• Maintains fluid distribution in and
albumin outside cells and plasma volume
 Half-life in plasma: 20 days
 Decreases rapidly in injury, • 80% of plasma oncotic pressure is
infection and surgery maintained by albumin
Functions : Fakulti Pergigian
• A non-specific carrier of
• hormones, calcium, free fatty acids,
drugs, etc.
• Tissue cells can take up albumin

by pinocytosis where it is
hydrolyzed to amino acids
• Useful in treatment of liver

diseases, hemorrhage, shock and
burns
: Fakulti Pergigian
: Fakulti Pergigian
Hypoalbuminemia
• Causes
• Decreased albumin synthesis (liver cirrhosis, malnutrition)
• Increased losses of albumin
• Increased catabolism in infections
• Excessive excretion by the kidneys (nephrotic syndrome)
• Excessive loss in bowel
• Severe burns (plasma loss in the absence of skin barrier)
Hypoalbuminemia : Fakulti Pergigian
Effects
• Edema due to low oncotic pressure
• Albumin level drops in liver disease causing low
oncotic pressure
• Fluid moves into the interstitial spaces causing
edema
• Reduced transport of drugs and other substances in
plasma
• Reduced protein-bound calcium
• Total plasma calcium level drops
• Ionized calcium level may remain normal
Hyperalbuminemia : Fakulti Pergigian
• No clinical conditions are known that cause

the liver to produce large amounts of albumin
• The only cause of hyperalbuminemia is
dehydration
Hypo-proteinemia
: Fakulti Pergigian
• Liver failure
• Nephrotic syndrome
• Malnutrition
• Malabsorption
• Severe burns
• Infection (↑catabolism)
• Genetic
: Fakulti Pergigian
a1-Antitrypsin
 Synthesized by the liver and macrophages
 An acute-phase protein that inhibits proteases
 Proteases are produced endogenously and from
leukocytes and bacteria
 Digestive enzymes (trypsin, chymotrypsin)
 Other proteases (elastase, thrombin)
 Infection leads to protease release from bacteria and
from leukocytes
Types of a1-Antitrypsin
: Fakulti Pergigian
• Over 30 types are known

• The most common is M type
• Genetic deficiency of a1-Antitrypsin
• Synthesis of the defective a1-Antitrypsin occurs in the
liver but it cannot secrete the protein
• a1-Antitrypsin accumulates in hepatocytes and is
deficient in plasma
Clinical Consequences of a1-Antitrypsin : Fakulti Pergigian
Deficiency
• Neonatal jaundice with evidence of cholestasis
• Childhood liver cirrhosis
• Pulmonary emphysema in young adults
Laboratory Diagnosis
• Lack of a1-globulin band in protein electrophoresis
• Quantitative measurement of a1-Antitrypsin by:

• Radial immunodiffusion, isoelectric focusing or nephelometry
: Fakulti Pergigian
a-Fetoprotein (AFP)
• Synthesized in the developing embryo and fetus by
the parenchymal cells of the liver
• AFP levels decrease gradually during intra-uterine
life and reach adult levels at birth
• Function is unknown but it may protect fetus from
immunologic attack by the mother
• No known physiological function in adults
: Fakulti Pergigian
a-Fetoprotein (AFP)
• Elevated maternal AFP levels are associated with:
• Neural tube defect, anencephaly
• Decreased maternal AFP levels are associated with:
• Increased risk of Down’s syndrome
• AFP is a tumor marker for:
Hepatoma and testicular cancer
: Fakulti Pergigian
Ceruloplasmin
• Synthesized by the liver
• Contains >90% of serum copper
• An oxidoreductase that inactivates ROS causing
tissue damage in acute phase response
• Important for iron absorption from the intestine
• Wilson’s disease:
• Due to low plasma levels of ceruloplasmin
• Copper is accumulated in the liver and brain
: Fakulti Pergigian
Haptoglobin
• Synthesized by the liver
 Binds to free hemoglobin to form complexes that

are metabolized in the RES
 Limits iron losses by preventing Hb loss from kidneys
 Plasma level decreases during hemolysis
: Fakulti Pergigian
Transferrin
 A major iron-transport protein in plasma
 30% saturated with iron
 Plasma level drops in:

 Malnutrition, liver disease, inflammation,
malignancy
 Iron deficiency results in increased hepatic

synthesis
 A negative acute phase protein

: Fakulti Pergigian
2–Microglobulin
• A component of human leukocyte antigen (HLA)
 Present on the surface of lymphocytes and most
nucleated cells
 Filtered by the renal glomeruli due to its small size
but most (>99%) is reabsorbed
 Elevated serum levels are found in
 Impaired kidney function
 Overproduction in disease
 May be a tumor marker for:
 Leukemia, lymphomas, multiple myeloma
: Fakulti Pergigian
C-Reactive Protein (CRP)

• An acute-phase protein synthesized by the liver
• Important for phagocytosis
• High plasma levels are found in many inflammatory

conditions such as rheumatoid arthritis
• A marker for ischemic heart disease
: Fakulti Pergigian
Hypergammaglobulinemia
• May result from stimulation of
• B cells (Polyclonal hypergammaglobulinemia)
• Monoclonal proliferation (Paraproteinemia)
Polyclonal hypergammaglobulinemia:
• Stimulation of many clones of B cells produce a wide
range of antibodies
• -globulin band appears large in electophoresis
• Clinical conditions: acute and chronic infections,
autoimmune diseases, chronic liver diseases
: Fakulti Pergigian
Monoclonal Hypergammaglobulinemia
 Proliferation of a single B-cell clone produces a single type of Ig

 Appears as a separate dense band (paraprotein or M band) in
electrophoresis
 Paraproteins are characteristic of malignant B-cell proliferation
 Clinical condition: multiple myeloma
: Fakulti Pergigian
Measurement of Plasma Proteins

A) Quantitativemeasurement of a
specific protein:
Chemical or immunological reactions
B) Semiquantitative measurement by
electrophoresis:
 Protes are separated by their electrical
charge in electrophoresis
 Five separate bands of proteins are
observed
 These bands change in disease
Methods of plasma protein separation : Fakulti Pergigian
Common methods of protein separation into:

albumin, globulins (alpha, beta & gamma) and fibrinogen by –
▪ Electrophoresis
▪ Salting out
▪ Ultracentrifugation
▪ Affinity chromatography
▪ Fractional precipitation method
▪ Immune electrophoresis
SLO 5. Explain the basic principles of Electrophoresis through
experiment : Fakulti Pergigian
Normal Pattern of Plasma Protein Electrophoresis
: Fakulti Pergigian
: Fakulti Pergigian
: Fakulti Pergigian
: Fakulti Pergigian
: Fakulti Pergigian
: Fakulti Pergigian
Positive Acute Phase Proteins

• Plasma protein levels increase in:
• Infection, inflammation , malignancy, trauma, surgery
• These proteins are called acute phase reactants

• Synthesized due to body’s response to injury
• Examples: a1-Antitypsin, haptoglobin,
ceruloplasmin, fibrinogen, c-reactive protein
: Fakulti Pergigian
Positive Acute Phase Proteins

Mediators cause these proteins to increase after
injury
Mediators: Cytokines (IL-1, IL-6), tumor necrosis

factors a and  , interferons, platelet activating
factor
Functions:
1. Bind to polysaccharides in bacterial walls
2. Activate complement system
3. Stimulate phagocytosis
: Fakulti Pergigian
Negative Acute Phase Proteins
• These proteins decrease in inflammation

• Albumin, prealbumin, transferrin
• Mediated by inflammatory response via cytokines
and hormones
• Synthesis of these proteins decrease to save amino
acids for positive acute phase proteins
Agarose Gel Electrophoresis : Fakulti Pergigian
Gel electrophoresis is a widely used technique for the analysis of

nucleic acids and proteins. Agarose gel electrophoresis is
routinely used for the preparation and analysis of DNA.
Gel electrophoresis is a procedure that separates molecules

on the basis of their rate of movement through a gel under
the influence of an electrical field.
We will be using agarose gel electrophoresis to determine the

presence and size of PCR products. PCR products indicate the
presence of Wolbachia.
• DNA is negatively charged.
• When placed in an electrical field, DNA will migrate toward the positive : Fakulti Pergigian
pole (anode).
• An agarose gel is used to slow the movement of DNA and separate by size.
H O2
 
DNA
- +
Power • Polymerized agarose is porous,

allowing for the movement of DNA
Scanning Electron Micrograph of
12/5/2019 Biochem/copps;dee
Agarose Gel (1×1 µm)  44
How fast will the DNA migrate?
: Fakulti Pergigian
strength of the electrical field, buffer, density of agarose gel…

Size of the DNA!
*Small DNA move faster than large DNA
…gel electrophoresis separates DNA according to size
DNA
small
large
- +
Power
Within an agarose gel, linear DNA migrate inversely
12/5/2019 proportionalBiochem/copps;dee
to the log10 of their molecular weight. 45
Agarose
: Fakulti Pergigian
D-galactose 3,6-anhydro
L-galactose
•Sweetened agarose gels have been

eaten in the Far East since the 17th
century.
•Agarose was first used in biology

when Robert Koch* used it as a
culture medium for Tuberculosis
bacteria in 1882
*Lina Hesse, technician and illustrator for a colleague of Koch was

the first to suggest agar for use in culturing bacteria
12/5/2019
Agarose is a linear polymer extracted from seaweed.
Biochem/copps;dee 46
: Fakulti Pergigian
Buffer
Making an Agarose Gel
An agarose gel is prepared by combining
agarose powder and a buffer solution.
Flask for boiling
Agarose
Electrophoresis Equipment : Fakulti Pergigian
Power supply
Gel tank Cover
Electrical leads

Casting tray Gel combs
: Fakulti Pergigian
Gel casting tray & combs
Preparing the Casting Tray
: Fakulti Pergigian
Seal the edges of the casting tray and put in the combs. Place the casting tray on
a level surface. None of the gel combs should be touching the surface of the
casting tray.
: Fakulti Pergigian
Agarose Buffer Solution
Combine the agarose powder and buffer solution. Use a flask that is
several times larger than the volume of buffer.
Melting the Agarose
: Fakulti Pergigian
Agarose is insoluble at room temperature (left).

The agarose solution is boiled until clear (right).
Gently swirl the solution periodically when heating to allow all the grains of agarose to
dissolve.
***Be careful when boiling - the agarose solution may become superheated and may boil
12/5/2019
violently if it has been heated too long in a microwave oven.
Biochem/copps;dee 52
Pouring the gel
: Fakulti Pergigian
Allow the agarose solution to cool slightly (~60ºC) and then

carefully pour the melted agarose solution into the casting tray.
Avoid air bubbles.
: Fakulti Pergigian
Each of the gel combs should be submerged in the melted agarose solution.
: Fakulti Pergigian
When cooled, the agarose polymerizes, forming a flexible gel. It should appear
lighter in color when completely cooled (30-45 minutes). Carefully remove the
12/5/2019 combs and tape. Biochem/copps;dee 55
: Fakulti Pergigian
Place the gel in the electrophoresis chamber.
: Fakulti Pergigian
DNA
buffer     
wells
Anode
Cathode (positive)
(negative)
Add enough electrophoresis buffer to cover the gel to a depth of at

least 1 mm. Make sure each well is filled with buffer.
Sample Preparation
: Fakulti Pergigian
Mix the samples of DNA with the 6X

sample loading buffer (w/ tracking
dye). This allows the samples to be
seen when loading onto the gel, and
increases the density of the samples,
causing them to sink into the gel
wells.
6X Loading Buffer: 
 Bromophenol Blue (for color)
 Glycerol (for weight)
Loading the Gel
: Fakulti Pergigian
Carefully place the pipette tip over a well and gently expel the sample. The
sample should sink into the well. Be careful not to puncture the gel with the
pipette tip.
Running the Gel
: Fakulti Pergigian
Place the cover on the electrophoresis chamber, connecting the electrical leads.
Connect the electrical leads to the power supply. Be sure the leads are attached
correctly - DNA migrates toward the anode (red). When the power is turned on,
bubbles should form on the electrodes in the electrophoresis chamber.
Cathode : Fakulti Pergigian
(-)
 wells
DNA  Bromophenol Blue
(-)

Gel
Anode
(+)
After the current is applied, make sure the Gel is running in the correct
direction. Bromophenol blue will run in the same direction as the DNA.
DNA Ladder Standard
: Fakulti Pergigian
-
 12,000 bp
 5,000
DNA
migration  2,000
 1,650
Note: bromophenol
blue migrates at  1,000
approximately the same  850
rate as a 300 bp DNA  650
molecule  500
 400
bromophenol blue  300
 200
+  100
Inclusion of a DNA ladder (DNAs of know sizes) on the gel makes it easy to determine the
sizes of unknown DNAs.
As an alternative to purchasing costly DNA ladders, one can be created using
meal worm (Tenebrio molitor) DNA and a restriction enzyme. : Fakulti Pergigian
http://people.uis.edu/rmosh1/DNAexerciseVIIa02.pdf

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: Fakulti Pergigian

Ts. RD.AIDIFITRINA BINTI KHIROTDIN

Principal College, Students Affairs

Centre of PreClinical Science Studies,

STUDENT LEARNING OUTCOME:

2. Name the major types of plasma protein

3. List the major role of plasma proteins

4. Name the example of disease that use plasma protein as

5. Explain the basic principles of Electrophoresis through

Albumins - 60%  Plasma contains >300 different proteins

▪ Liver - 90% plasma proteins

Liver dysfunction - plasma proteins

Types of Plasma Proteins

• Transport (Albumin, prealbumin, globulins)

1. Colloid osmotic pressure in blood Colloid osmotic

Colloid osmotic pressure (oncotic pressure) and filtration of fluid

• Brain: Cerebral edema

12/5/2019 Facial oedema Biochem/copps;dee 10

• Most abundant plasma protein • Maintains oncotic pressure:

• Tissue cells can take up albumin

• Useful in treatment of liver

• No clinical conditions are known that cause

• Over 30 types are known

• Quantitative measurement of a1-Antitrypsin by:

 Binds to free hemoglobin to form complexes that

 Limits iron losses by preventing Hb loss from kidneys

 Plasma level decreases during hemolysis

 Plasma level drops in:

 Iron deficiency results in increased hepatic

 A negative acute phase protein

C-Reactive Protein (CRP)

• Important for phagocytosis

• High plasma levels are found in many inflammatory

• A marker for ischemic heart disease

 Proliferation of a single B-cell clone produces a single type of Ig

Measurement of Plasma Proteins

Common methods of protein separation into:

Normal Pattern of Plasma Protein Electrophoresis

Positive Acute Phase Proteins

• These proteins are called acute phase reactants

Positive Acute Phase Proteins

Mediators: Cytokines (IL-1, IL-6), tumor necrosis

Negative Acute Phase Proteins

• These proteins decrease in inflammation

Gel electrophoresis is a widely used technique for the analysis of

Gel electrophoresis is a procedure that separates molecules

We will be using agarose gel electrophoresis to determine the

Power • Polymerized agarose is porous,

strength of the electrical field, buffer, density of agarose gel…

•Sweetened agarose gels have been

•Agarose was first used in biology

*Lina Hesse, technician and illustrator for a colleague of Koch was

Flask for boiling

Gel tank Cover

Casting tray Gel combs

Gel casting tray & combs

Agarose Buffer Solution

Agarose is insoluble at room temperature (left).

Allow the agarose solution to cool slightly (~60ºC) and then

Place the gel in the electrophoresis chamber.

Add enough electrophoresis buffer to cover the gel to a depth of at

Mix the samples of DNA with the 6X