Plasma Proteins

Immunoglobulins
Plasma Proteins
1. Blood is a very important fluid
• What is blood?

• What does blood do?

 Blood - Functions

• Respiratory
– Transport O2 from lungs to tissues
– Transport CO2 from tissues to lungs
• Nutrition
– Transport “food” from gut to tissues (cells)
• Excretory
– Transport waste from tissues to kidney
(urea, uric acid, water)
• Regulatory
– Water Content of Tissues
• Water exchanged through vessel walls to tissue

• Body Temperature
• Protective
– Antibodies, antitoxins, white blood cells
(WBC)
• Acid-base balance
– pH 7.35~7.45, NaHCO3/H2CO3
• Coagulation
• Blood composition
– 70 mL/kg of body weight
– 5 L (average) in an adult
– Suspension of cells in a carrier fluid (plasma)
• Cells - 45% by volume
• Plasma - 55% by volume
• Cells
– Red cells (erythrocytes):
• 5x106/L
– White cells (leukocytes)
• 7x103/L
– Platelets (thrombocytes)
• 3x105/L
 Add anticoagulants
Centrifuged Blood Sample (heparin,
potassium oxalate)
 Separation of Components

Plasma = Less Dense

Platelets / WBCs

RBCs
More Dense
 2. Plasma vs. serum
•Plasma is the Serum is the liquid p
liquid, cell-free art of blood AFTER c
part of blood, that oagulation, therfore
has been treated devoid of clotting fac
with anti- tors as fibrinogen.
coagulants.

Anticoagulated Clotted

•serum= plasma - fibrinogen

 Components of Plasma
Blood plasma Consists of:
• Water 90%
• Plasma Proteins 6-8 %
• Electrolytes (Na+ & Cl-) 1%

Other components:
• Nutrients (e.g. Glucose and amino acids)
• Hormones (e.g. Cortisol, thyroxine)
• Wastes (e.g. Urea)
• Blood gases (e.g. CO2, O2)
 3. Plasma proteins

※ A large number of dissolved proteins of

the plasma
※ includes
※ simple proteins, conjugated proteins
※ carry out a number of different
functions.
  Separation
1. Electrophoresis 2. Ultra-centrifuge

A/G=1.5~2.5
Plasma proteins
General characteristics of plasma proteins
1. They are synthesized in liver except immuno
globulin.
2. Almost all plasma proteins are glycoproteins.
3. Many plasma proteins exhibit polymorphism s
uch as α1-antitrypsin, transferrin, haptoglobin.
4. Each plasma protein has a characteristic hal
f-life in the circulation.
5. Acute Phase Proteins, APP
 3.1 Albumin

• Albumin (69 kDa), single polypeptide chain having

585 aa with 17 disulfide bonds, is the most abund
ant protein (60%) in the blood plasma. (3.5-5.0 g/
dl)

• Synthesis of albumin:
– Liver produced about 12g albumin per day which represent 2
5% of total hepatic protein synthesis and 50% of secreted
protein. half-life: 20 days
– For this reason, measurement of serum albumin concentratio
n is used to assays liver function test.
 Human Serum Albumin

 Structure
1. Shape: ellipsoid.
2. Charge: pI=4.0.
3. Domain: three major domains.
  Function
1. Transport: It can bind and transport many diverse mol
ecules and serve as low-specificity transport protein, w
hich include:
– a. Metal ions: such as calcium and copper.
– b. Free fatty acid: albumin binds to free fatty acid releas
ed by adipose tissue and facilitates their transfer to othe
r tissue.
– c. Bilirubin: this protects from the toxic side effects of u
nconjugated bilirubin.
– d. Bile acid: albumin carries the bile acids that are recycl
e from the intestine to the liver in the hepatic portal vein.
– e. Hormones: such as thyroid hormones and the steroid ho
rmones.
 2. Maintain of osmotic pressure
• Colloid osmotic pressure, is a form of osmotic pres
sure exerted by proteins in blood plasma that usua
lly tends to pull water into the circulatory system.
– Because large plasma proteins cannot easily cross th
rough the capillary walls.
• In conditions where plasma proteins are reduced,
– e.g. from being lost in the urine (proteinuria) or fro
m malnutrition,
– there will be a reduction in osmotic pressure, leading
to enhanced fluid retention in tissue spaces (edema).
 Colloid osmotic pressure

Low albumin, causing edema.

 Clinical aspects
1. Albumin binds different drugs
and strongly affects the phar
macokinetics of these drugs.
 For example, sulfonamides c
an cause the release of unconj
ugated bilirubin from albumin
by competitive binding. If give
n to infants, sulfonamides may
lead to kernicterus.
2. In cases of liver disease or st
arvation, albumin synthesis dec
reases.
 This lead to edema.
 Clinical aspects
3. Hypoalbuminemia
– lowered plasma albumin
– in malnutrition, nephrotic syndrome and cirrhosi
s of liver.
4. Albuminuria
– albumin is excreted into urine
– in nephrotic syndrome and certain inflammatory
conditions of urinary tract.
5. Albumin is therapeutically useful for the t
reatment of burns and hemorrhage.
3.2 Globulins
 3.2.1 α1–Antitrypsin
/α1–Antiproteinase(α1–AT or AAT)

It (52 kDa) is a glycoprotein with 394 aa.

It is a major constituent of α1 globulin fracti
on of plasma protein, normal concentration abo
ut 200mg/dl.
It is a serine protease inhibitor and can c
ombines with trypsin, elastase and other prote
ase and inhibits them.
  Clinical significance
1. Emphysema: used to represent the
abnormal distension of lungs.
– About 5% is due to the deficiency o
f α1–AT.
– This is associated with lung infection
and increase the activity of macrop
hage to release elastase that damag
e lungs tissue.

 Smoking can cause oxidation of Met358 to

methionine sulfoxide and inactivate α1–AT.
2. α1 –antitrypsin
deficiency liver disease
 due to mutant α1 –
antitrypsin
accumulates and
aggregates to form
polymers, by unknown
mechanism, cause liver
damage followed by
accumulation of
collagen resulting in
fibrosis (cirrhosis).
 3.2.2 α2 –Macroglobulin
(α2 –MG)

It (720 kDa) is a glycoprotein with 4 identica

l subunits, a major constituent of α2 fraction.
It is a panprotease inhibitor and can comb
ine and inhibit many protease.
It can bind cytokines such as PDGF and TGFβ
and target them to particular cells to affect o
n cell growth or function.
 Clinical significance
 α2 -MG levels are increased in nephrotic sy
ndrome
– a condition wherein the kidneys start to leak
out some of the smaller blood proteins. Becau
se of its size, α2 -MG is retained in the blood
stream.
• This increase has little adverse effect on th
e health, but is used as a diagnostic clue.

normal nephrotic syndrome

 3.2.3 Hepatoglobin (H
p)
 It (90 kDa) is a glycoprotein.
 It can bind with the free hemoglobin (extra-corpuscula
r Hb) in a tight noncovalent complex Hp-Hb during hemol
ysis.
 Hp-Hb(155 kDa) cannot pass through glomeruli of kidne
y while free Hb(65kDa) can and Hp prevent the loss o
f free Hb into urine.

※Low levels of plasma concentration of Hp can diagnos

e hemolytic anemia.
t1/2 of Hp: 5 day, Hp-Hb: 90 min.
 3.2.4 Ceruloplasmin(CER)
It (160 kDa) is a blue-coloured, copper-con
taining α2 fraction.
It can carry 90% of plasma copper tightly s
o that copper is not readily exchangeable. It
processes copper-dependent oxidase activity.
Albumin carries the other 10% , which is th
e major supplier of copper to tissue.
 Clinical significance
Low level of ceruloplasmin is associated with Wi
lson’s disease (hepatolenticular degeneration)
 Wilson's disease is an inherited disorder in which t
here is too much copper in the body's tissues. The
excess copper damages the liver and nervous s
ystem .
Treatment: penicillamine is the first treatment
used.
 This binds copper (chelation) and leads to excretion
of copper in the urine.
3.2.5 Transferrin (T
f)
It (76 kDa) is a glycoprotein, part of β fra
ction.
It can transport iron in plasma as ferric ion
s (Fe3+) and protect the body against the toxi
c effects of free iron.
 3.2.6 Acute Phase Proteins, APP
 The levels of certain plasma proteins change during
inflammation, infection, injury, cancer etc. These protei
ns are “Acute Phase Proteins, APP ”
 Include C-reactive protein,CRP, α1-acid glycoprotein,
fibrinogen, haptoglobin ,α1-antitrypsin, albumin and transf
errin.
 APP are believed to play a role in the body’s respon
se to inflammation, changes in their plasma concentratio
ns are generally regarded as being sensitive, although no
n-specific, indicators if inflammation.
 C-reactive protein, CRP
• A major component of acute phase prote
in.
• It reacts with the C polysaccharide of pn
eumococci.
• Involved in the promotion of immune sys
tem through the activation of compleme
nt cascade.
• Estimation of CRP in serum is important Complement
activation
for the evaluation of acute phase respon
se.
– CRP rises up to 50,000-fold in acute inflamm
ation, such as infection. It rises above normal
limits within 6 hours, and peaks at 48 hours.
Complement: rupturing
membranes of foreign
cells, cell swells and
Immunoglobulins
※ Immunoglobulin(Ig)/
antibody(Ab):
※ Glycoprotein molecules that are pro
duced by plasma cells in response t albumin
o an immunogen and which function
as antibodies, mostly associated wit

Amount of
h γ fraction. globulins

protein
※ But γ-globulin and Ig are not synon
ymous.
※ Ig is a functional term
※ γ-globulin is physical term. - +
Mobility
  General Functions of Immunoglobulin
1. Antigen(Ag) binding
- Ig binds to a specific antigenic determinant
2. Effector functions
- Complement activation
- Binding to various cells such as phagocytic c
ells, lymphocytes, mast cells: antibody-medi
ated phagocytosis or antibody-dependent cell
-mediated cytotoxicity (ADCC).
 Two Forms of Ig
1. Membrane Ig, mIg
• It confers antigenic specificity on
B cells.

mIg
2. Secreted Ig, SIg
It can circulate in the blood and s
erve as the effectors of humoral im
munity by searching out and neutrali
zing antigens or marking them for el
imination.
SIg
 Basic Structure
1. four chains (H2L2): Y shape
• two identical light chains
(L): 23 kDa
• two identical heavy chain
s (H): 53-75 kDa
2. Disulfide bonds and such non
covalent interactions as salt
linkages, hydrogen bonds an
d hydrophobic bonds to form
heterodimer (H-L).
 1. Variable region (V):
VL&VH
2. Constant region (C):
CL&CH
3. Hinge region:
flexibility
Hinge • Light Chain:
region
– VL (110 aa) + CL (110 a
a)
• Heavy Chain:
– VH (110 aa) + CH1-CH3 (or
CH4) (330-440 aa)
Structural Regions
CH3
CH2

CH3
CH1

CH2

CH3
VH1
CH1

CH2

CH3
VH1
CH1

VL

CH2

CH3
VH1
CH1

VL CL

CH2

CH3
VH1
CH1

VL CL

CH2

CH3
 VH1
CH1

CL VL

CH2
Elbow

Hinge

CH3
Enzymatic Digestion Products of
Immunoglobulins
 Immunoglobulin Fragments:
Structure/Function Relationships
Papain is a sulfhydryl p
rotease from Carica
papaya latex

• Fab Papain

– Ag binding
– Valence = 1
– Specificity deter
mined by VH and
VL
• Fc ( crystallizable) Fc
– Effector functions
Fab
F(ab’)2: a single fragment composed of two F
ab-like fragments, it is divalent.
Hydrolytic fragment of Immunoglobulin
 • Functions of the domains on Ig

Ag Binding

Complement Binding Site

Binding to Fc
Receptors
Placental Transfer
• Hypervariable re
gion:
also called Co
mplementarity D
etermining Regio
ns (CDRs)
 Antigenic determinant or epitope:
The structure recognized by an antibody

Concept: Epitopes can bind in pockets or grooves or on ex

tended surfaces in the binding site of antibodies.
 Immunoglobulin Classes and Subclasses

• In terms of the differences in amino acid sequen

ce of constant region of heavy chain, immunglobu
lin molecules are divided into 5 classes:
– IgG, IgA, IgM, IgD and IgE
• Heavy chain:
– 5 types: γ,α,μ,δ and ε.
• Light chains
– 2 types: κand λ.
 Immunoglobulin Classes of Mammals

1. IgG - γ heavy chains

2. IgM - µ heavy chains
• pentamer
3. IgA - α heavy chains
• dimer
4. IgD - δ heavy chains
5. IgE - ε heavy chains

monomer dimer pentamer

   IgG

IgG1, IgG2, IgG4 IgG3

 It is the most abundant class in serum, constitut
es about 80% of the total serum Ig.
 4 subclasses, IgG1, IgG2, IgG3, and IgG4.
 All IgG's are monomers. The subclasses differ in
the number of disulfide bonds and length of the hin
ge region.
  Functions of IgG
1. Major Ig in extravascular spaces.
2. Placental transfer: IgG is the only clas
s of Ig that crosses the placenta.
3. Complement activation.
4. Binding to cells - Macrophages, monocyte
s, PMNs (polymorphonuclear leukocyte), and s
ome lymphocytes have Fc receptors for the
Fc region of IgG.
 Fc receptor
• Fc receptor: protein found on the surface
of certain cells (including natural killer ce
lls, macrophages, neutrophils, and mast cel
ls).
• Fc receptors bind to antibodies that are a
ttached to infected cells or invading path
ogens.
• Their activity stimulates phagocytic or cy
totoxic cells to destroy microbes, or infec
ted cells by antibody-mediated phagocyto
sis or antibody-dependent cell-mediated c
ytotoxicity.
 IgA
• Structure
– Serum - monomer
– Secretions (sIgA)
• Dimer
• J chain
• Secretory component

Secretory Piece J Chain

 IgA
• Function
– 2nd highest serum Ig
– Major secretory Ig (Mucosal or Local Immunit
y)
• Found in the body secretions: tears, breast milk,
saliva, mucus of the bronchial, genitourinary, a
nd digestive tract
• IgA is the most predominant antibody in the col
ostrum, the initial secretion from the mother’ b
reast after a baby is born.
– Does not activate complement (unless aggre
gated)
– Binds to Fc receptors on some cells
  IgM
Structure
• The largest Ig compo
sed of 5 Y-shaped un
its held together by
a J polypeptide chain.
1. Pentamer
2. Extra domain (CH4)
3. J chain
  Functions of IgM
1. 3rd highest serum Ig.
2. IgM cannot traverse blood vessels, hence it
is restricted to the blood stream.
3. 1st Ig produced in a primary response to an
antigen and serve as first line of defense.
4. a good complement activation Ig. Thus, IgM
is the most effective in leading to the lysis
of microorganisms.
5. Binds to Fc receptors.
 IgD

• Structure
– Monomer
– Tail piece

Tail Piece
 IgD
• Properties
– 4th highest serum Ig, its role in serum
uncertain.
– B cell surface Ig.
– Does not bind complement.
 IgE
• Structure
– Monomer
– Extra domain
(CH4)

CH4
 IgE
• Function
– Least common serum Ig
– Allergic reactions
Binds to basophils and mast cells (Does
not require Ag binding)
– Parasitic infections (Helminths)
• Binds to Fc receptor on eosinophils
– no complement activation.
 Points
• Plasma vs. serum
• Components and functions of Plasma
• Plasma proteins
– Albumin: Function(Transport, Maintain of
osmotic pressure), Clinical aspects
– Globulins
• α1–Antitrypsin, α2 –Macroglobulin, Hepatoglobin (Hp),
Ceruloplasmin, Transferrin (Tf)
– Acute Phase Proteins(APP), C-reactive protein
(CRP)
• Immunoglobulin(Ig)/antibody(Ab)
– General Functions, Forms, Basic Structure, Enzymatic Dig
estion Products (Fab,Fc, F(ab’)2)
– Classes: IgG, IgM, IgA, IgD, IgE (Structure and Function)