UNIVERSITY Of JORDAN . . .
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LECTURE NO: 1
;--1 DATE: 14/ r I 2(10 g' I
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PlaSlTIa Proteins And IrnlTIunoglobulins
- Plasma is the fluid part of blood after separation of red blood cells.
- Plasma contains variety of constituents including electrolytes,
nutrients, metabolites, and waste products.
- Plasma proteins have many functions but the most important role is
the osmotic pressure: these proteins are too large to cross the
endothelial membranes.
- The fact that these proteins do not leave blood vessels allows
distrjbution & movement of water between plasma and intercellular
fluid.
If the level of these proteins decreased in plasma water will not return
back to blood causing edema in the tissue spaces.
- The plasma proteins are mixture of proteins including simple proteins
that is formed only from amino acids with no other groups, conjugated
proteins which contain other non amino acid part (ex: glycoproteins),
enzymes, hormones, transport proteins, and blood clotting proteins
... etc
- Some plasma proteins are secreted to do their functions in the plasma
& some are found ill plasma due to turnover of the cell (death of the
cell)
- A total protein in the plasma is 7.0-7.5 gldL (70-75 giL) therefore the
major solid part of the plasma is proteins.
Methods for Separation of Proteins
1- Depending on solubility (salting out): proteins have different
solubility in the presence of salts. If we add salt and increase its
concentration in plasma some proteins will precipitate, other will
remain soluble. So we can isolate proteins according to their
different degrees of solubility.
2- Electrophoresis: migration of the charged molecules under
electrical field.
• The more the charge the faster the migration.
• The larger the mass the slower the migration.
• So this method depends on both mass & charge.
• This method uses (I) electrical field. (2) Buffer: in order to maintain
certain charges on proteins. (3) Supporting media like cellulose
acetate.
• At pH> 7 the protein is negatively charged, migrate toward the
positive electrode.
• At pH < 7 the protein is positively charged, migrate toward the
negative electrode.
• When proteins migrate to opposite electrodes this forms different
pads on the graph according to their specific rate of migration.
• Because these proteins are mostly colorless we use special stain to
react with the proteins presenting them in the graph.
• To measure the quantity of proteins in plasma we .use a device called
Densemeter that measures the density of the color of the stain. the
area under the peak corresponds to quantity, Albumin has the
highest peak and so the highest percentage in plasma.
• Globulins are of four types: alpha 1, alpha 2, beta, & gamma.
3- Isoelectric focusing: powerful way to separate proteins by their
isoelectric points using pH gradient. Proteins have a different
isoclectric point which is the pH where the protein has zero net
charge and at which the protein would stop migrating in the
field and could be isolated.
4- Using Antibodies: immunoglobulins that react specifically with
certain proteins.
Origin of Plasma Proteins
Most of them from LIVER
- Some by endothelial cells
- & some by plasma cells which are derived from white blood cells (8
cells).
Synthesis of the liver can be proofed by variety of techniques:
I. Isolated perfused liver: by measuring quantity of proteins in the
hepatic vein compared that in arteries that go to the liver.
2. Liver slices: by taking a liver from an animal and making slices ofit,
we can see the proteins that are synthesized. This method is done
using radioactive amino acid like radioactive glycine.
3. ·Using Liver homogenates: liver slices in which there is no longer
intact cells.
4. We can isolate mRNA from liver cells, then put it in invitro system
which contain all amino acids and other components needed for
protein synthesis.
Synthesis as PreProteins
'" The synthesis of plasma proteins occurs in rough endoplasmic
membrane associated with bound ribosomes.
- Proteins that arc going to be exported outside the cell begin with
signal sequence that led the protein to the lumen of the rER.
'" Then in the smooth endoplasmic reticulum the PreProtein undergoes
modification which could be proteolysis or addition of carbohydrates....
'" Then modified further in Goigi complex and transported in secretory
vesicles and finally releases from the cell by EXQcytosis.
- Proteins that function in the cytosol are synthesized on free ribosomes
in cytosol.
Polymorph ism
'" Polymorphism is different amino acid sequence in different
individuals that performs normal functions. If the different amino acid
sequence do abnormal function this is called mutation.
• It is genetically determined.
• Some proteins that exhibit polymorphism are:
Alphal antitrypsin, haptoglobin, transferrin.
Half Life
It is the time required to degrade half of the proteins during their own
turnover
It is variable: in Haptoglobin it is 5 days. however in Albumin it is 20
days.
Half life is decreased as a result of certain diseases. Inflammation of
the G.I tube, for example, might be accompanied by losing proteins
causing shorter half lives. This phenomenon is known as Protein
Loosing Gastroenteropathy.
How can we study a protein half life? Simple. take protein sample
from an animal & label it with radioactive Iodine that will bind to
proteins without affecting its function. Then inject the labeled sample
back to the animal, after some time take a sample and observe the
level of radioactivity which corresponds to the quantity of the non
degraded proteins; so half life is measured by quantity of protein after
different time intervals.
Acute Phase Proteins
These are the proteins that increase in level in certain acute
inflammatory states, and increase in certain types of tissue damage,
also in chronic inflammatory states and in cancer. They are useful in
diagnosis of certain diseases.
Example of acute phase proteins:
1) C- reactive protein (CRP)
2) alpha 1 antitrypsin
3) Haptoglobin
4) Alphal glycoprotein
5) Fibrinogen
*** Interleukin I
• It stimulates the synthesis of the majority of acute phase proteins
• It is produced by mononuclear phagocytic cells.
Albumin
.:. 60% 0 f total protein
·:·3.4-4.7g/dL
.:. It is just one single type of protein, however globulins are
mixture of several proteins
.;. 12 grams of albumin are produced daily in the liver.
.:. Synthesized early as PreProProtein which is modified by
removing the signal sequence and hexapeptide ( certain 6 amino
acids) to form the albumin protein.
•:. Albumin is so big, it has huge mass = 69 kDa (585 amino acids):
the mass is calculated by multiplying 585 ( no. of amino acids in
albumin molecule) times 110 (avg. amino acid mass in Daltons)
= 69000 Daltons.
•:. It is one polypeptide chain
.:. Contain 17 disulfide bonds
.:. Responsible for 75 - 80% of osmotic pressure
.:. Low level of albumin cause edema
.:. Responsible for binding to variety of ligands, some are water
insoluble.
Albumin is general carrier molecule of water insoluble substances like FFA
(free fatty acids), steroid hormones,
.:. It also binds Calcium ions: the physiological activity depends on the
free (unbound) Calcium, but we in the lab measure the total Calcium
that equals free + bound. Calcium and other ligands when bind to
albumin become inactive. So the level of albumin affect both bound
and totals Calcium but not free Calcium.
•:. Albumin binds Bilirubin whkh is a substance propuced by
degradation of Heme.
•:. It also binds variety of drugs.
•:. Drug-Dnlg lnteractions: when albumin binds drugl and we add drug2
that also become bound to albumin, drug2 competes and replaces
drug 1 causing more drug I to be released as free ligand; that will result
in increase in the activity & perfonnance of drugl above normal,
sometimes towards a toxic level.
THE END
