Biochem 01

UNIVERSITY Of JORDAN . . .

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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 - -

I. 2.

3. 4.

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: Isolated perfused liver: by measuring quantity of proteins in the hepatic vein compared that in arteries that go to the liver. 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. ·Using Liver homogenates: liver slices in which there is no longer intact cells. 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

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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