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PLASMA PROTEINS
Questions:
1. Plasma proteins Dec1992
2. Electrophoresis- April 2001; oct 1999; Feb 2007;
3. Chromotography - March 2002; April 95 and 99
4. Electroporesis of serum proteins- April 1994
5. Electroporesis and its applications aug 2006
6. Partion chromatography (principle and clinical application)
7. Thin layer chromatography
8. Functins of immunoglobulins
9. Wilsons disease Nov 1998; March 2002
10.Structure of immunoglobins Dec 1992
11.Detoxificaion of xenobiotics.April 2001
12.Antioxidants - Dec 2005
13.Write a note on serum electroporesis. Pon May 2010
14.Write the structure of immunoglobins. Nov 2010
15.Draw a labeled diagram of a typical immunoglobin. Pon May 2013
16.Give normal levels of serum proteins (total, albumin,and globulin) Pon Nov
2007
Plasma:
Plasma is blood minus formed elements like WBC, RBC etc; Blood volume is 4.5 to 5
litres. Plasma volume is 2.5 to 3 litres; The packed cell volume or hematocrit is about
45%. The term serum is applied to the liquid mediumw hich separateos ut aftert he
blood clots (coagulates). Serum is plasma minus fibrin. All except immunoglobulins are
synthesized in liver. Most plasma proteins are glycoproteins. The gamma globulins are
produced by cells of the body's immune system.
Plasma proteins values:
Total: 6 to 8 gm/dl.
Albumin: 3.5 to 5 gm/dl
Globulin: 2.5 to 3.5 gm/dl.
Fibrinogen: 200 to 400 mg/dl
Separation of plasma proteins:
1. Salting out process: In laboratory, separation can be done by salts. Thus, fibrinogen
is precipitated by 10% and globulins by 22% concentration of sodium sulphate.
Ammonium sulphate will precipitate globulins at half saturation and albumin at full
saturation.
2. In clinical laboratory, total proteins in serum or plasma of patients are estimated by
Biuret
method.
3. Albumin is quantitated by Bromo cresol green (BCG) method, in which the dye is
preferentially bound with albumin, and the color is estimated colorimetrically.
4. Electrophoresis:
a. The term electrophoresis refers to the movement of charged particles through
an electrolyte when subjectedv to an electric field.
b. In agar gel electrophoresis serum is separated into 5 bands. The concentration
of each one of these fractions can be estimated by a densitometer as follows:
1. Serum albumin
5565 %
2. Alpha-1 globulins
2-4 %
3. Alpha-2 globulins
6-12 %
4. Beta globulins
8-12 %
5. Gamma globulins
12-22 %
c. Normal electrophoretic pattern:

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c. The relative
proportions of plasma
proteins can vary in certain diseases and electrophoretic tracings showing such
changes can be a useful diagnostic aid.
d. Abnormal electrophoretic pattern Electrophoresis of serum proteins is conveniently
used for the diagnosis of certain diseases
1. Multiple myeloma : A sharp and distinct M band appears in the globulin
fraction.
2. Acute infections : a1- and a2- globulins are increased.
3. Nephrotic syndrome: Decreaseda lbumin with sharp and prominent a 2globulin.
4. Primary immune deficiency : Diminished globulin band.
5. a1-Antitrypsind eficiency: Diminishedc a1- globulin band.
e. Albumin/globulin (A/G) ratio: The albumin concentration of plasma is 3.5 to 5.0
g/dl while that of total globulins is 2.5 to 3.5 g/dl. The normal A/G ratio is 1.2 to
1.5 : 1. The A/G ratio is lowered either due to decrease in albumin or increase in
globulins, as found in the following conditions:
1. Decreased synthesis of albumin by liver usually found in liver diseases and
severe
protein malnutrition.
2. Excretion of albumin into urine in kidney damage.
3. Increased production of globulins associated with chronic infections,
multiple myelomas etc.
Components of plasma proteins:
Albumin:
1. Albumin is a polypeptide with 585 amino acids; synthesized in liver; 1/2 life is 20
days; present in vascular, CSF and interstitial fluids;
2. Functions:
1. Exerts colloid osmotic pressure which is responsible for maintaining blood
volume; in hypoalbuminaemia interstitial water seepage leads to edema.
2. It acts as carrier for Bilirubin, drugs, hormones and metals like Ca, Cu, etc.
3. It has buffering action to maintain blood pH
4. It serves as source for tissue nutrition
3. Clinical significance:
1. Albumin bound substances cannot cross blood brain barrier; free Bilirubin
crosses it to cause kernicterus.
2. Albumin binding causes drug interaction one replacing the other and
enhancing its toxicity.
3. Ca is bound to albumin and also resent in free ionic forms
4. Albumin is helpful to treat burns and shock states.
5. Hypoalbuminaemia occurs in cirrhosis liver, malnutrition, albuminuria etc.
6. Albulin globulin ratio is reversed in conditions of hypoalbuminaemia and
hyperimmuno globulinaemia
Acute phase reactants:
1. The level of certain proteins in blood may increase 50 to 1000 folds in various
inflammatory and neoplastic conditions. Such proteins are acute phase proteins.
2. C-Reactive Protein (CRP):
i. So named because it reacts with C-polysaccharide of capsule of

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pneumococci. CRP is a beta-globulin and is synthesized in liver in response
to inflammation.
ii. It stimulates complement activity and macrophage phagocytosis during
inflammation.
iii. When the inflammation has subsided, CRP quickly falls, followed later by
ESR (erythrocyte sedimentation rate).
iv. CRP level, especially high sensitivity C-reactive protein level in blood has a
positive correlation in predicting the risk of coronary artery diseases.
v. Normal value: < 1 mg/dl.
3. Ceruloplasmin
1. Ceruloplasmin is blue in color (Latin, caeruleus = blue). It is an alpha-2
globulin in liver lymphocytes and macrophages. After the formation of
peptide part (apo-Cp) copper is added by an intracellular ATPase and
carbohydrate side chains are added to make it a glycoprotein (holo-Cp).
2. Ceruloplasmin is also called Ferroxidase, an enzyme which helps in the
incorporation of iron into transferrin. It is an important anti-oxidant in
plasma.
3. Ninety per cent of copper content of plasma is with ceruloplasmin, and 10%
with albumin. Copper is bound with albumin loosely, and so easily
exchanged with tissues. Hence transport protein for copper is Albumin.
4. Lowered level of ceruloplasmin is seen in Wilson's disease, malnutrition,
nephrosis, and cirrhosis.
5. Ceruloplasmin is an acute phase protein. Increased plasma Cp levels are
seen in active hepatitis, biliary cirrhosis, hemochromatosis, and obstructive
biliary disease, pregnancy, estrogen therapy, inflammatory conditions,
collagen disorders and in malignancies.
6. Drugs increasing the ceruloplasmin level are estrogen and contraceptives.
7. Reference blood levels of ceruloplasmin are:
1. Adults Males- 22-40 mg/dl
2. Females 25-60 mg/dl
3. Females on oral contraceptives: 27-66 mg/dl
4. Pregnancy 30-120 mg/dl
8. Wilson's Disease
1. Level is reduced to less than 20 mg/dl in Wilson's hepatolenticular
degeneration. It is an inherited autosomal recessive condition. Incidence
of the disease is 1 in 50,000.
2. The basic defect is a mutation in a gene encoding a copper binding
ATPase in cells, which is required for excretion of copper from cells. So,
copper is not excreted through bile, and hence copper toxicity. Please
also see Chapter 35, under copper metabolism.
3. Increased copper content in hepatocyte inhibits the incorporation of
copper to apo-ceruloplasmin. So ceruloplasmin level in blood is
decreased.
4. Clinical features
i. Accumulation in liver leads to hepatocellular degeneration and
cirrhosis.
ii. Deposit in brain basal ganglia leads to lenticular degeneration
and neurological symptoms.
iii. Copper deposits as green or golden pigmented ring around
cornea; this is called KayserFleischer ring.
iv. Treatment consists of a diet containing low copper and
injection of D-penicillamine which excretes
5. Haptoglobin (Hp)

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1. is a plasma glycoprotein with an approximate molecular weight of
90,000. Hp is an acute phase protein since its plasma concentration is
increased in several i nflammatory conditions.Haptoglobin binds with
the free hemoglobin (known as extra-corpuscularh emoglobin)
thatspills into the plasma due to hemolysis. The haptoglobi nhemoglobin (H p-Hb) complex (mol. wt. 155,000) cannot pass through
glomeruli of kidney while free Hb (mol. wt. 65,000) can. Haptoglobin,
therefore, prevents the loss of free Hb into urine.
2. Clinical significance of Hp : Hemolytic anemia is associated with
decreased plasma concentration of haptoglobin. This is explained as
follows. The haltt-life of Hp is about 5 days while that of Hp-Hb
complex is 90 min. ln hemolytic anemia, free Hb in plasma is elevated
leading to increased formation of Hp-Hbcomplex. This complex, in
turn, is rapidly cleared from the plasma resulting in decreased Hp
levels.
Negative Acute Phase Proteins
1. During an inflammatory response, some proteins are seen to be decreased in blood;
those are called negative acute phase proteins.
2. Examples are albumin, transthyretin (pre-albumin), retinol binding protein and
transferrin.
3. Transferrin is a specific iron binding protein. It has a half-life of 7-10 days and is used
as a better index of protein turnover than albumin.
Alpha-1 Anti-trypsin (AAT)
1. It is otherwise called alpha-anti-proteinase or protease inhibitor. It inhibits all serine
proteases (proteolytic enzymes having a serine at their active center), such as
plasmin, thrombin, trypsin, chymotrypsin, elastase, and cathepsin.
2. Serine protease inhibitors are abbreviated as Serpins.
3. AAT is synthesized in liver. It is a glycoprotein with a normal serum level of 75-200
mg/dl.
4. AAT deficiency causes the following conditions:
i. Emphysema: The incidence of AAT deficiency is 1 in 1000, and is one of the
commonest inborn errors. The total activity of alpha1-AT is reduced in these
individuals. Bacterial infections in lung attract macrophages which release
elastase. In the alpha 1-AT deficiency, unopposed action of elastase will cause
damage to lung tissue, leading to emphysema. About 5% of emphysema cases
are due to alpha1-AT deficiency.
ii. Nephrotic syndrome: AAT molecules are lost in urine, and so AAT deficiency is
produced.
iii. Alpha1-Antitrypsin deficiency and liver disease : This is due to the
accumulation of a mutant
iv. a1- AT which aggregates to form polymers. These polymers,in turn-by an
unknown mechanismcause liver damage (hepatitis) followed by accumulation
of collagen resulting in fibrosis (cirrhosis).
Transport proteins
1. Blood is a watery medium; so lipids and lipid soluble substances will not easily
dissolve in the aqueous medium of blood. Hence, such molecules are carried by
specific carrier proteins.
2. Albumin: It carries bilirubin, free fatty acids, calcium and drugs.
3. Pre-albumin or Transthyretin: it carries thyroid hormones, thyroxin (T4) and tri-iodo
thyronine (T3).
4. Retinol binding protein (RBP): It carries vitamin A. It is a low molecular weight

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protein, and so is liable to be lost in urine. To prevent this loss, RBP is attached
with prealbumin; the complex is big and will not pass through kidney glomeruli. It
is a negative acute phase protein.
5. Thyroxine binding globulin (TBG): It is the specific carrier molecule for thyroxine
and tri-iodo
thyronine. TBG level is increased in pregnancy; but decreased in nephrotic
syndrome.
6. Transcortin: It is also known as Cortisol binding globulin (CBG). It is the transport
protein for
cortisol and corticosterone.
7. Haptoglobin: Haptoglobin (for hemoglobin), Hemopexin (for heme) and Transferrin
(for iron)
are important to prevent loss of iron from body.
Clotting Factors:
i. The coagulation factors are present in circulation as inactive zymogen forms. They
are converted to their active forms only when the clotting process is initiated. This
would prevent unnecessary intravascular coagulation. Activation process leads to a
cascade amplification effect, in which one molecule of preceding factor activates
1000 molecules of the next factor.
ii. Several of these factors require calcium for their activation. The calcium ions are
chelated by the gamma carboxyl group of glutamic acid residues of the factors,
prothrombin, VII, IX, X, XI and XII The gamma carboxylation of glutamic acid residues
is dependent on vitamin K.
Clinical Applications of albumin:
1. Blood brain barrier: Albumin bound bilirubin does not cross blood brain barrier.
Free unconjugated bilirubin can cross bloodbrain barrier and get deposited in
brain. The brains of young children are susceptible; free bilirubin deposited in brain
leads to kernicterus and mental retardation.
2. Drug interactions: When two drugs having high affinity to albumin are
administered together, there may be competition for the available sites, with
consequent displacement of one drug. Such an effect may lead to clinically
significant drug interactions, e.g. phenytoin dicoumarol interaction.
3. Calcium level in blood is lowered in hypoalbuminemia. Thus, even though total
calcium level in blood is lowered, ionized calcium level may be normal, and so
tetany may not occur.
4. Human albumin is therapeutically useful to treat burns, hemorrhage and shock.
5. Hypo-albuminemia will result in tissue edema (Starling's law).
a. Malnutrition, where albumin synthesis is depressed (generalized
edema)
b. Nephrotic syndrome, where albumin is lost through urine (facial
edema)
c. Cirrhosis of liver (mainly ascites), where albumin synthesis is less and it
escapes into ascitic fluid.
d. Chronic congestive cardiac failure: Venous congestion will cause
increased hydrostatic pressure and decreased return of water into
capillaries and so pitting edema of feet may result.
6. Normal value: Normal level of Albumin is 3.55 g/dl. Lowered level of albumin
(hypo-albuminemia) has important clinical significance.
IMMUNOGLOBULINS (S.N)

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1. Immunoglobulins are glycoprotein molecules that are produced by plasma cells in
response to an immunogen and which function as antibodies. Immunoglobulins produced
by B-lymphocytes.
2. General functions of immunoglobulins:
a. Immunoglobulins protect the body against bacterial and viral infections.
b. Immunoglobulins bind specifically to one or a few closely related antigens. Antigen
binding by antibodies results in protection of the host.
c. Effector Functions : The binding of an antibody to an antigen leads to certain
"effector functions" of antibodies. Such effector functions include:
i. Fixation of complement - This results in lysis of cells and release of biologically
active molecules
ii. 2. Binding to various cell types - Phagocytic cells, lymphocytes, platelets, mast
cells, and basophils have receptors that bind immunoglobulins. This binding
can activate the cells to perform some immune function.
3. Basic structure of immunoglobulins
a. Although different immunoglobulins can differ structurally, they all are built from the
same basic units. All immune
globulins have a four chain structure as their basic
unit. They are composed of two identical light chains and two identical heavy
chains . The heavy and light chains and the two heavy chains are held together by
interchain disulfide bonds. Within each of the polypeptide chains there are also intrachain disulfide bonds.
b. Both the heavy and light chain could be divided into two regions based on variability
in the amino acid sequences. These are the:
1. Light Chain - 214 amino acids
2. Heavy Chain - 440 amino acids
c. The immunoglobulins can be divided into five different classes, based on differences
in the amino acid sequences in the constant region of the heavy chains.
1. IgG - Gamma heavy chains
2. IgM - Mu heavy chains
3. IgA - Alpha heavy chains
4. IgD - Delta heavy chains
5. IgE - Epsilon heavy chains

d. Variable (V) and Constant (C) Regions

The heavy and light chain could be divided into two regions based on variability in

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the amino acid sequences. These are called V L and CL and VH and CH regions.
e. Hinge Region
This is the region at which the arms of the antibody molecule forms a Y. It is called
the hinge region because there is some flexibility in the molecule at this point.
f. Fab and Fc portions: Digestion with papain breaks the immunoglobulin molecule in
the hinge region. This results in the formation of two identical fragments that contain
the light chain and a portion of heavy chain. These fragments were called the Fab
fragments because they contained the antigen binding sites of the antibody.
g. Digestion with papain also produces a fragment that contains the remainder of the
two heavy chains. This fragment was called Fc because it was easily crystallized. Fc
portion is the site for complement fixation.
COLLAGEN
Short notes: structure of Collagen - april 99
1. It is a structural protein. 25-30% of body protein is collagen; major fibrous element
of bones, teeth, tendons, cartilage and blood vessels.
2. Structure:
a. The tropocollagen or "collagen molecule" is a subunit of larger collagen
aggregates such as fibrils. It is approximately 300 nm long and 1.5 nm in
diameter, made up of three polypeptide strands (called alpha peptides),
each possessing the conformation of a left-handed helix. These three lefthanded helices are twisted together into a right-handed coiled coil, a triple
helix or "super helix", a cooperative quaternary structure stabilized by
numerous hydrogen bonds. The sequence often follows the pattern Gly-Pro-Y
or Gly-X-Hyp, where X and Y may be any of various other amino acid
residues.
3. Synthesis: It is synthesized by fibroblasts. Inside fibroblast polypeptides are
synthesized proline and lysine residues are hydroxylated and glycosylation of lysine
takes place. The procollagen is secreted as a precursor and is cleaved by specific
peptidases to form tropocollagen. Tropocollagen is assembled into
collagen.Collagen solution on heating forms gelatin.
4. Functions:
a. Support to organs
b. Alignment and anchoring of cells.
c. It is part of blood vessel walls and and when exposed initiates thrombus
formation.
5. Abnormalities:
a. Ontogenesis imperfecta: Autosomal dominant; defets in triple helix
formation; brittle bones and skeletal deformities.
b. Ehlers - Danlos syndrome: defective type II collagen formation; loose skin
and hypermobile joints.
c. Homocystinuria: homocysteine reacts with lysyl adehydes to block cross
linking . Vascular and skeletal deformities and ocular defects.
d. Marfans syndrome: defect in synthesis of fibrillin.
e. Vit C deficiency leads to weak collagen fragile vessels.
f. Lathyrism: lathyrus sativa poison inhibits lysyl oxidase and leads to defective
collagen formation.
Albumin-Globulin Ratio
In hypo-albuminemia, there will be a compensatory increase in globulins which are
synthesized by the reticulo-endothelial system. Albumin-globulin ratio (A/G ratio) is thus
altered or even reversed. This again leads to edema.

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Hyper-gamma-globulinemias
i. Low albumin level: When albumin level is decreased, body tries to compensate by
increasing the production of globulins from reticulo-endothelial system.
ii. Chronic infections: Gamma globulins are increased, but the increase is smooth and
wide based
iii. Multiple myeloma:
1. Drastic increase in globulins is seen in paraproteinemias, when a sharp spike
is noted in electrophoresis. This is termed as M-band because of the
monoclonal origin of immunoglobulins. The monoclonal origin of
immunoglobulins is seen in multiple myeloma.
2. Monoclonal gammopathies are characterized by the presence of a
monoclonalprotein which can be detected by serum protein electrophoresis
and typed by immunofixation. electrophoresis.
3. The light chains are produced in excess which is excreted in urine as Bence
Jones proteins (BJP) when their serum level increases. Multiple myeloma is the
most common type of monoclonal gammopathy. Free light chain assay along
with kappa and lambda ratio in serum and urine is found to be very useful in
early diagnosis, monitoring the response to treatment and prediction of
prognosis.