8/14/2019 Biochem 01

1/6

, ~

UNIVERSITY Of JORDAN ..._ ~ ~ E _ - a ~ C _ u _ l t _ Y ~ O _ F _ M ~ e _ d _ i c _ i n _ e _ ~ _ '

rLECTURE NO: 1-

;--1 DATE: 14/ r I 2(10 g' IDONE BY: ;v(0 h C{ 1(I/J1 eelWe( e..-e...

' - lOR.: ~ L Khatl/J

8/14/2019 Biochem 01

2/6

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 theendothelial membranes.

- The fact that these proteins do not leave blood vessels allowsdistrjbution & movement of water between plasma and intercellularfluid.If the level of these proteins decreased in plasma water will not returnback to blood causing edema in the tissue spaces.

- The plasma proteins are mixture of proteins including simple proteinsthat is formed only from amino acids with no other groups, conjugatedproteins 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 thecell)

- A total protein in the plasma is 7.0-7.5 gldL (70-75 giL) therefore themajor solid part of the plasma is proteins.

Methods for Separation of Proteins1- Depending on solubility (sal ting out): proteins have differentsolubility in the presence of salts. Ifwe add salt and increase itsconcentration in plasma some proteins will precipitate, other willremain soluble. So we can isolate proteins according to theirdifferent degrees of solubility.2- Electrophoresis: migration of the charged molecules underelectrical 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 maintaincertain charges on proteins. (3) Supporting media like cellulose

acetate.

8/14/2019 Biochem 01

3/6

At pH> 7 the protein is negatively charged, migrate toward thepositive electrode.

At pH < 7 the protein is positively charged, migrate toward thenegative 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. thearea under the peak corresponds to quantity, Albumin has thehighest 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 differentisoclectric point which is the pH where the protein has zero netcharge and at which the protein would stop migrating in thefield and could be isolated.

4- Using Antibodies: immunoglobulins that react specifically withcertain proteins.

Origin of Plasma ProteinsMost 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 thehepatic vein compared that in arteries that go to the liver.

2. Liver slices: by taking a liver from an animal and making slices o fit,we can see the proteins that are synthesized. This method is doneusing radioactive amino acid like radioactive glycine.

3. Using Liver homogenates: liver slices in which there is no longerintact cells.

4. We can isolate mRNA from liver cells, then put it in invitro systemwhich contain all amino acids and other components needed forprotein synthesis.

8/14/2019 Biochem 01

4/6

Synthesis as PreProteins'" The synthesis of plasma proteins occurs in rough endoplasmicmembrane associated with bound ribosomes.- Proteins that arc going to be exported outside the cell begin withsignal sequence that led the protein to the lumen of the rER.'" Then in the smooth endoplasmic reticulum the PreProtein undergoesmodification 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 ribosomesin cytosol.

Polymorph ism'" Polymorphism is different amino acid sequence in differentindividuals that performs normal functions. If the different amino acidsequence do abnormal function this is called mutation. It is genetically determined. Some proteins that exhibit polymorphism are:Alphal antitrypsin, haptoglobin, transferrin.

Half LifeIt is the time required to degrade halfof the proteins during their ownturnoverIt is variable: in Haptoglobin it is 5 days. however in Albumin it is 20days.Half life is decreased as a result of certain diseases. Inflammation ofthe G.I tube, for example, might be accompanied by losing proteinscausing shorter half lives. This phenomenon is known as ProteinLoosing Gastroenteropathy.How can we study a protein half life? Simple. take protein samplefrom an animal & label it with radioactive Iodine that will bind toproteins without affecting its function. Then inject the labeled sampleback to the animal, after some time take a sample and observe the

8/14/2019 Biochem 01

5/6

level of radioactivity which corresponds to the quantity of the nondegraded proteins; so half life is measured by quantity of protein afterdifferent time intervals.

Acute Phase ProteinsThese are the proteins that increase in level in certain acuteinflammatory states, and increase in certain types of tissue damage,also in chronic inflammatory states and in cancer. They are useful indiagnosis of certain diseases.Example of acute phase proteins:1) C- reactive protein (CRP)2) alpha 1 antitrypsin3) Haptoglobin4) Alphal glycoprotein5) 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 aminoacids) 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 inalbumin 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

8/14/2019 Biochem 01

6/6

.:. Low level of albumin cause edema

.:. Responsible for binding to variety of ligands, some are waterinsoluble.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 thefree (unbound) Calcium, but we in the lab measure the total Calcium

that equals free +bound. Calcium and other ligands when bind toalbumin become inactive. So the level of albumin affect both boundand totals Calcium but not free Calcium.:. Albumin binds Bilirubin whkh is a substance propuced bydegradation 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 replacesdrug1 causing more drug I to be released as free ligand; that will resultin increase in the activity & perfonnance of drugl above normal,sometimes towards a toxic level.

THE END