Blood (Plasma, Serum) Proteins

Objectives
Know the major plasma proteins and where they are

produced.
Describe the main properties and functions of some

key plasma proteins.

Give a detailed explanation of the role of at least two

plasma proteins in disease and clinical diagnosis

Blood
When cells are removed

Plasma Water (90%) blood proteins (8%) inorganic electrolytes salts, lipids glucose

remove fibrinogens/clotting factors (clot) serum

All bodily fluids have some proteins:

Plasma (3-8g/dL)

lymphatic fluids

cerebrospinal fluid (45mg/dL).

Urine (trace amounts)

Examples of plasma proteins:

Albumin, Clotting factors, Haptoglobin Transferrin, Ceruloplasmin Lipoproteins -antitrypsin -macroglobulin, Immunoglobulins The complement system.

Plasma 2DE proteomics with and without depletion of high abundance plasma proteins by affinity removal

How are plasma proteins categorised?

Simple proteins:

Albumin

Conjugated Proteins:
Glycoproteins, Lipoproteins

Size
small globulins (40kda)
macroglobulins (1Mda) lipoproteins (20-40 Mda

The salting-out method produces three groups:

Fibrinogen
Albumin Globulins

Separation
Cellulose acetate electrophoresis
Gels (agarose) five bands Albumin, 1, 2, , and quantified by densitometry

Figure 1. The image on the left is pure serum. The image on the seperation of serum into different categories when the serum goes through electrophoresis

 http://www.youtube.com/watch?v=z2D7ZkT3aOo

Five groups on cellulose acetate or agarose:

Albumin (quantitatively the largest protein).
1-globulins (mainly 1-antitrypsin). 2-globulins (2-macroglobulin and haptoglobin,

immunoglobulins).

 -globulins ( two types 1; transferrin & LDL, 2 the

C3 component of complement, immunoglobulins).

a fibrinogen band will be seen if plasma is used. -globulins (immunoglobulins).

The Electrophoresis pattern of plasma proteins in disease

The Electrophoresis pattern of plasma proteins in disease

 Reduction of all proteins in malnutrition

(maladsorption)

Acute phase pattern (increases in acute phase

proteins associated with inflammation; 1- and 2globulins and an increase in plasma viscosity)

Chronic inflammation increased immunoglobulins

especially -globulins, but the pattern is complicated depending on cause.

 Cirrhosis of the liver (raised -globulins and reduced

albumin and 1-globulins)

Nephrotic syndrome (reduced albumin and -

globulins, there may be increases in 2macroglobulin

1-antitrypsin deficiency decreased 1-antitrypsin

 Paraproteinaemia (a dense abnormal band on

electrophoresis strip, can be suggestive of malignancy)

hypogammaglobulinaemia

Classification by function
Immunoglobulins
Clotting factors Enzymes Transporters

What determines the level of Plasma Proteins ?

Synthesis

Catabolism (excretion)
Tissue distribution

Most major plasma proteins are synthesised by a few cell types:

Hepatocytes, (albumin, clotting factors, transferrin)

Macrophages
B cells (-globulins) Low abundance proteins?

 Other important characteristics Plasma Proteins exhibit polymorphism

Almost all are glycosylated (N- or O-linked

oligosaccharide chains).
Removal of the sugars alters function and

half-life
Excessive glycosylation seen in which

disease?

The circulation half-life is important , why?

Albumin
Haptoglobin

20 days
5 days

altered by certain diseases.

In Crohns disease (regional ileitis)

Inflamed

intestinal mucosa

loss of plasma proteins into the bowel

albumin half-life to as little as 1 day.

Plasma proteins levels may also increase following:

Cancer
Chronic inflammation In acute Inflammation

 Several Plasma proteins increase in response to

trauma.
"acute phase proteins:
Levels increase in the plasma following acute

inflammation, trauma

Examples of acute phase proteins

C-reactive protein
alpha-1 anti-chymotrypsin alpha-1 anti-trypsin

haptoglobins
caeruloplasmin serum amyloid A fibrinogen ferritin complement components C3, C4

 The level of some plasma proteins may decrease : pre-albumin albumin transferrin

 synthesis of acute phase proteins by hepatocytes

triggered by (cytokines) lnterleukins (IL-l, IL-6),

released from mononuclear phagocytic cells

C-reactive protein ( reacts with the C polysaccharide

of pneumococci),

 1-antitrypsin haptoglobin, 1-acid glycoprotein Fibrinogen. The increase may range from a 2-l000-fold.

Why elevated ?
They mediate the response to inflammation. Stimulation of complement pathway (CRP), neutralize proteases released during the acute

inflammatory state (-AT).

 Acute-phase proteins

trapping of microorganism and their products, activating the complement system, binding cellular remnants (nuclear fractions) neutralizing enzymes, scavenging free hemoglobin and radicals modulating the hosts immune response.

The functions of plasma

proteins
Functionally plasma

proteins can be placed in 7 categories

1. The distribution of fluid between blood and tissues.

They exert osmotic pressure (oncotic pressure), approximately 25 mm Hg.

[Plasma proteins] (severe protein malnutrition, liver disease),

oncotic pressure of plasma

fluid accumulates in the extravascular tissue spaces, a condition known as oedema.

2.

Carriers for cations and insoluble compounds, fatty acids, billirubin, steroids, lipids, vitamins. Drugs The immune system:

3.

Antibodies (gamma globulins), also complement, acute phase proteins.

4. Hormones (quantitatively small, fetoprotein)

5.Clotting factors (quantitatively small) 6. Source of energy and amino acids

7.

Enzymes (intracellular from breakdown of cells, acetylcholinesterase)

THE PLASMA PROTEINS

Albumin
The major plasma protein
Albumin (69 kDa) is the major plasma protein (3.4-

4.7 g/dL)
> 60% of the total plasma proteins.

40% in the plasma,

60% in the extracellular space.

Produced by the liver (12g/day)

25% of total hepatic protein synthesis
Synthesised as a preproprotein In some diseases the synthesis of albumin is

reduced decreased albumin/globulin ratio

 Decreased albumin synthesis is an

early indicator of protein malnutrition (kwashiorkor).

Analbuminia: individuals lack albumin

(mutation),

Other proteins may be increased to compensate

Albumin is responsible for 80% of the osmotic

pressure of plasma

Analbuminia may result in mild oedema

Changes in plasma proteins may

Affect binding of various ions in the blood
pharmacokinetics of certain drugs

Structure:
one polypeptide chain of 585 amino acids and

contains 17 disulphide bonds.

3 domains may be identified using proteases,

Despite its high concentration it does not increase the viscosity of the plasma.
A significant part of the transport role

of plasma is carried out by albumin.

Binding
fatty acids calcium, amino acids, bilirubin steroid hormones drugs.

Transferrin
A glycoprotein
-globulin

Molecular mass 76 kDa.

Synthesized in the liver. 20 polymorphic forms

What does transferrin do?

A central role in iron transport (2 mol of Fe3+ per mole

of transferrin)
Iron (Fe2+ ) ingested at the intestinal mucosa is tightly

regulated.

 Plasma transferrin concentration is approximately

300 mg/dL
Capable of binding 300g/dL But normally only one-third saturated Very little iron is lost in healthy adult (1 mg/d) more

in adult females

RBC destruction

25mg/day released (potentially toxic)

Binding to transferrin (

toxicity)

What happens to iron-bound transferrin?

Transferrin cell surface receptor binds the protein The receptor is internalized by endocytosis The iron released (to ferritin) Receptor returns to the surface

Problems of Iron Metabolism:

Particularly important for two groups:

Women Older people

Iron deficiency (anaemia) due to:

inadequate intake
inadequate utilisation, excessive loss

 In terms of diagnosis transferrin is used

for?

Low iron intake

Ferritin
binds and stores 23% iron in the body

in tissues such as: liver spleen.

Ceruloplasmin
2-globulin Copper binding protein (90% of plasma copper, each

molecule binds six atoms of copper very tightly.)

About 160 kDa. Plasma levels are reduced in liver disease.

Low levels in:

Wilson disease

abnormal metabolism of copper.

1-Antiproteinase (1-Antitrypsin)
The principal serine protease inhibitor of human

plasma (serpin).
As name suggests first discovered as 1-Antitrypsin Controls the proteolytic action of lysosomal enzymes.

 Synthesized in hepatocytes and macrophages

But aslo intestinal and bronchial epithelial cells

About 52 kDa
A single-chain protein of 394 amino acids,

Contains three oligosaccharide chains,

The major component (> 90% ) of the 1 fraction of human

plasma.

Levels increase after infection/trauma

How does it work?

Complexes with proteins causing inhibition of:

Trypsin Elastase (other proteases)

Highly polymorphic ( > 70 forms) identified by

electrophoresis.

 The basic clinical problem is due to the mutations in

the 1-Antitrypsin gene

Highly polymorphic ( > 70 forms) identified by

electrophoresis. More by PCR

 Autosomal codominant
Major genotype is MM (phenotypic; PiM) on

chromosome 14
There is also the Z allele and the S allele Most individuals have two copies of the M allele (MM) Any deviation from this may result in either liver

disease or lung disease (emphysema).

 With the ZZ (PiZ) genotype

Less protein is secreted from cells this can lead to tissue

damage.

PiZZ results from a single point mutation at position 342

on the gene encoding the substitution of lysine for glutamate (Glu342Lys).

 The PiZ mutant is responsible for more than

95% of cases of pulmonary and hepatic disease associated antititrypsin deficiency,

Another common mutant is the PiS, Individuals may have two of these abnormal genes

labelled PiSS or PiZZ, or one of each PiSZ

 Accumulation of the PiZ protein in the ER of

hepatocytes is seen in some liver diseases

The PiZ mutations produce a peptide which forms

polymers in the rough endoplasmic reticulum of hepatocytes,these aggregate and are retained in the liver.
This may lead to hepatitis or cirrhosis (accumulation

of massive amounts of collagen, resulting in fibrosis) of the liver.

Lung tissue damage and 1-antitrypsin deficiency

How does 1-antitrypsin deficiency result in tissue

damage ?

Normal 1-antitrypsin expression and level:

Phagocyte in lung

Active elastase + 1-AT

Tissue damage

Inactive elastase:1-AT complex

No proteolysis of lung (no tissue damage)

Abnormal 1-antitrypsin expression and level:

Active elastase (no 1-antitrypsin)

Proteolysis of lung

Tissue damage

 Smoking results in the oxidation of a methionine

residues in the protein decreasing binding.

In patients with the PiZZ phenotype

The effect is exaggerated accelerating the the emphysema

2-Macroglobulin
a large glycoprotein (720 kDa)

four identical subunits of 180kDa

10% of the plasma proteins transportation zinc in plasma (10%)

 The major member of a group of plasma proteins

called: thiol ester plasma protein family (complement proteins C3 and C4)
they contain a unique internal cyclic thiol ester bond

and a proteolytic cleavage region

These helps attract and deactivate protineases

Where are they synthesized?

Many cells synthesized the protein including: Monocytes Hepatocytes, Astrocytes.

What is the function of 2-macroglobulin

To neutralize a range of Proteinases

To bind and targets certain cytokines to tissues.