My plasma proteins


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My plasma proteins

  2. 2. Learning Objectives • To know different type of plasma proteins present in human body. • Their distribution and functions • Methods of separation and estimation • To know each protein in greater depth • Clinical importance of the proteins • Diseases associated with these proteins
  3. 3. Plasma - blood treated with anticoagulants to prevent clot formation then centrifuged to remove the cells Plasma Plasma “Buffy coat” Buffy coat Red blood cells Red blood cells Serum - blood that is allowed to form a fibrin clot then centrifuged to remove the cells and the clot
  4. 4. The Composition of Whole Blood Figure 19.1b
  5. 5. Plasma proteins • include proteins of blood plasma and proteins of interstitial fluid • almost all are glycoproteins • some groups of proteins are classified separatelly (enzymes, proteohormones) • “total protein” ~ more than 300 proteins
  6. 6. Individual proteins of blood plasma The figure is from (Feb 2007)
  7. 7. Classification of plasma proteins • by electrophoretic mobility  prealbumins  albumin  alpha, beta and gamma-globulins  fibrinogen The figure is from textbook: Devlin, T. M. (editor): Textbook of Biochemistry with Clinical Correlations, 4th ed. Wiley-Liss, Inc., New York, 1997. ISBN 0-471-15451-2
  8. 8. The figure is from textbook: Devlin, T. M. (editor): Textbook of Biochemistry with Clinical Correlations, 4th ed. Wiley-Liss, Inc., New York, 1997. ISBN 0-471-15451-2
  9. 9. Principal proteins of each fraction immunoglobulins: IgG, IgA, IgM 2-macroglobulin haptoglobin 1-antitrypsin transferrin orosomucoid C3-complement
  10. 10. • by specific function  transport proteins  proteins of immune system  system of proteases and antiproteases  proteins of hemocoagulation system  signal proteins  enzymes  cellular proteins
  11. 11. • by clinical use  cardiomarkers  tumormarkers  acute phase reactants  cellular enzymes  hormones  cytokines
  12. 12. protein Mr (x 103) albumin 66 transferrin 80 haptoglobin 1-1 85 IgG 144 IgA 160 haptoglobin 2-2 160 2-macroglobulin IgM 720 971 (accepted from book: Clinical Laboratory Diagnostics / Lothar Thomas)
  13. 13. The plasma proteins are found into three major groups : • fibrinogen • albumin • globulins Relative dimension and approximate molecular masses of protein molecules in the blood
  14. 14. Some Functions of Plasma Proteins
  15. 15. General properties of plasma proteins  Most are synthesized in the liver exception: -globulins – synthesized in plasma cells  Synthesized as pre-proteins on membrane-bound polyribosomes; then they are subjected to posttranslational modifications in ER and Golgi apparatus  Almost all of them are glycoproteins Exception: albumin  They have characteristic half-life in the circulation (albumin – 20 days)   Many of them exhibit polymorphism (immunoglobulins, transferrin…)
  16. 16. Proteins of interstitial fluid • subcutaneous: albumin • lymph: less proteins than in plasma • liquor: 200x less than in plasma • pathological fluids:  transudate  exudate < 30 g/l > 30 g/l
  17. 17. Factors influencing concentration of proteins total protein: 64 – 83 g/l • rate of synthesis and degradation • distribution in body fluids • loss into the third space • elimination from the body • hydration of the body
  18. 18. other important factors: • elevation of concentration before taking blood sample  body position ( in supine position)  tightening of arm • storage of biological specimens
  19. 19. Physiological variability • increased concentrations     plasma > serum (fibrinogen) stand-up position (by 10-15 %) increased muscle activity (by 12 %) dehydration • decreased concentrations  children, pregnant women  after starvation (albumin, transferrin, C3)
  20. 20. Regulation of synthesis INCREASE        inflammation hyperthyroidism hypercortisolism Growth hormone iron deficiency protein loss clonal production of Ig DECREASE  liver damage with parenchymal tissue  nutritional deficit  hypothyroidism  diabetes mellitus  alcoholism
  21. 21. Reversible Equilibrium Between the Plasma Proteins and the Tissue Proteins.
  22. 22. Methods of separation • Precipitation by SALTING OUT: • Different concentration of salt solutions are used to precipitate different concentration of protein. • Salts most commonly used - Ammonium sulphate solution -Mixture of Sodium Sulphate and Sodium Sulphite  On 1/5th saturation with ammonium sulphate FIBRINOGEN is best precipitated.  On 1/3rd saturation globulin starts precipitating.It is called Euglobulin.
  23. 23.  On ½ saturation total globulin is precipitated out. It is called Pseudoglobulin.  Albumin is precipitated out by full saturation. • Fractionation of plasma proteins by ETHANOL [Cohn’s Fractionation] Varying concentration of ethanol at low temperature Cohn’s Fractions Fraction I rich in Fibrinogen Fraction II γ-globulin Fraction III α and β globulins Including isoaglutinnin & prothrombin Fraction IV α &β globulins Fraction V albumin
  24. 24. Advantage of this fractionation  Ethanol that is used for separation can be readily removed by evaporation.  Being a mild procedure it does not cause denaturation of protein. Clinically Cohn’s method is used for obtaining purified proteins on large scale for therapeutic purpose. ELECTROPHORESIS: Initially paper electrophoresis was used. Nowadays agar gel electrophoresis, cellulose acetate membrane electrophoresis, immunoelectrophoresis is used.
  25. 25. • Proteomic analysis: • • • • • Nephelometry Immunoturbimetry Gel based separation technique e.g. 2-D PAGE Mass spectrometry MALDI-TOF
  26. 26. Many Plasma Proteins Exhibit Polymorphism Human plasma proteins that exhibit polymorphism include      1- antitrypsin, haptoglobin, transferrin, ceruloplasmin, and immunoglobulins The polymorphic forms of these proteins can be distinguished by different procedures (eg, various types of electrophoresis or isoelectric focusing), in which each form may show a characteristic migration. Analysis of these human polymorphisms have proved to be of genetic, anthropologic, and clinical interest.
  27. 27. ALBUMIN  Albumin (69 kDa) is the major protein of human plasma.  Normal level is 3.5 - 5.5g/dL  Makes up approximately 60% of the total plasma protein.  About 40% of albumin is present in the plasma, and the other 60% is present in the extracellular space.  The liver produces about 12g of albumin per day.  Gene for albumin is situated at chromosome no. 4
  28. 28. Albumin is synthesized by hepatocytes as preproalbumin. Its signal peptide is removed, converting it to proalbumin. In turn, proalbumin, while inside transport vesicles, is converted to albumin by action of furin. Preproalbumin Signal Peptide Signal Peptide + Proalbumin Furin Hexapeptide + ALBUMIN Furin is a enzyme that cleaves a hexapeptide from proalbumin immediately C-terminal to a dibasic amino acid site (Arg). The resulting mature albumin is secreted into the plasma. The synthesis of albumin decreases relatively early in conditions of protein malnutrition, such as kwashiorkor. In liver diseases the synthesis of albumin is depressed (decrease in albumin/globulin ratio).
  29. 29. Structure and Functions of Albumin Mature human albumin consists of one polypeptide chain (simple protein) of 585 amino acids and contains 17 disulfide bonds. Albumin has an ellipsoidal shape, which means that it does not increase the viscocity of the plasma. Because of its relatively low molecular mass (about 69 kDa) and high concentration albumin is thought to be responsible for 70-80% of the osmotic pressure of human plasma. By the use of proteases, albumin can be subdivided into three domains, each have different functions. .
  30. 30. Functions of Albumin maintenance of colloidal oncotic pressure Transport of long chain fatty acids & sterols Transport of bilirubin Binding and solubilization of drugs
  31. 31. Hypoalbuminemia Low blood albumin levels (hypoalbuminemia) can be caused by: •Liver disease; cirrhosis of the liver is most common •Excess excretion by the kidneys (as in nephrotic syndrome) •Excess loss in bowel (protein-losing enteropathy) •Burns (plasma loss in the absence of skin barrier) •Redistribution (hemodilution [as in pregnancy]) •Acute disease states (referred to as a negative acutephase protein)
  32. 32. Hyperalbuminemia -Due to severe or chronic dehydration and high protein diets. - Chronic dehydration needs to be treated with zinc as well as with water. - Zinc reduces cell swelling caused by increased intake of water (hypotonicity) and also increases retention of salt. -In the dehydrated state, the body has too high in osmolarity and, it discards zinc to prevent this.
  33. 33. GLOBULINS • α-globulins: • Further classified into α1 and α2 globulin according to electrophoretic mobility. α1 acid glycoprotein:  Also called orosomucoid  Normal level 0.6 – 1.4 gm/lit  Carbohydrate content is about 41%  Considered as a reliable indicator of acute inflammation  binds to progesterone hormone and transport it.
  34. 34. • α- fetoprotein: • Present in high conc. in fetus particularly in mid pregnancy. • Normal adult level < 1µg /100ml. • Potential tumour marker of hepatocellular carcinoma. • Forms the basis of TRIPLE test of screening for DOWNS syndrome.
  35. 35. α1-ANTITRYPSIN ( 1-ANTIPROTEINASE) 1-Antitrypsin (about 52 kDa) is a single chain protein of 394 amino acids, contains three olygosaccharide chains. It is synthesized by hepatocytes and macrophages and is the principal serine-protease inhibitor (Serpin or Pi) of human plasma. Inhibits trypsin and elastase.
  36. 36. Deficiency of Antitrypsin-I At least 75 polymorphic forms occur, many of which can be seperated by electrophoresis. The major genotype is MM and its phenotypic product is PiM. A deficiency of this protein has a emphysema. This occurs in subjects with the ZZ genotype who syntyhesize PiZ and also in PiSZ heterozygotes. When the amount of 1-antitrypsin is deficient and polymorphonuclear white blood cells increases in the lung (eg, during pneumonia), the affected individual lacks a countercheck to a proteolytic damage of lung by proteases such as elastase.
  37. 37. Deficiency of Antitrypsin-II Methionine (358 residue) of to proteases. 1-antitrypsin is involved in its binding Smoking oxidizes this methionine to methionine sulfoxide and this results in proteolytic destruction of lung tissue (eg, PiZZ; already have a low levels of 1-antitrypsin). Deficiency of 1-antitrypsin is also implicated in one type of liver of liver disease (in ZZ type). Conformational diseases (Loop-sheet polymerization): Most appear to be due to the formation of β-sheets by conformationally unstable proteins, which in turn leads to formation of aggregates. At present, severe 1-antitrypsin deficiency liver disease can be successfully treated by liver transplantation.
  38. 38. CERULOPLASMIN [α-2 globulin]  Conc. in plasma: 300 mg l  It is a glycoprotein with enzyme activity ( copper oxidase, histaminase, ferrous oxidase )  Functions:  carries 90% of copper in plasma (copper – cofactor for a variety of enzymes); 1 molecule binds 8 atoms of copper; [½ as cuprous and ½ as cupric ] binds copper more tightly than albumin that carries other 10% of copper albumin may be more important in copper transport (donates copper to tissues more readily)
  39. 39. Causes of ceruloplasmin decrease:  Liver diseases, in particular Wilson´s disease:  genetic disease in which copper fails to be excreted into the bile and accumulates in liver, brain, kidney, and red blood cells  cause: mutations in the gene encoding for copper-binding ATPase  consequences: • accumulation of copper in liver, brain, kidneys… liver disease, neurologic symptoms • ↓ coupling of copper to apoceruloplasmin low plasma levels of ceruloplasmin Causes of ceruloplasmin increase:  Inflammatory states  Carcinomas, leukaemia  Rheumatoid arthritis
  40. 40. HAPTOGLOBIN (Hp) [α-2 globulin] Haptoglobin (Hp) is a plasma glycoprotein that binds extracorpuscular hemoglobin (Hb) in a tight noncovalent complex (Hb-Hp). Haptoglobin binds to extracorpuscular hemoglobin, preventing free hemoglobin from entering the kidney. Hb-Hp complex is too large to pass through the glomerulus. The function of Hp thus appears to be to prevent loss of free Hb into the kidney. Different fates of free hemoglobin and of the Hb-Hp complex
  41. 41. Polymorphic forms of Haptoglobin Human haptoglobulin exists in three polymorphic forms, known as Hp 1-1, Hp 21, and Hp 2-2. Hp1-1,is the simplest polymorphic form in human. Hp1-1migrates in starch gel electrophoresis as a single band, whereas Hp2-1and Hp2-2 exhibit much more complex patterns. It has been suggested that the haptoglobuin polymorphism may be associated with the prevalence of many inflammatory diseases.
  42. 42. Functions of Haptoglobin Free Hb passes through the glomerulus of the kidney, enters the tubules, and tends to precipitate therein. However the Hb-Hp complex is too large to pass through the glomerulus. The function of Hp thus appears to be to prevent loss of free Hb into the kidney. This conserves the valuable iron present in hemoglobin, which would otherwise be lost to the body.
  43. 43. Functions of Haptoglobin Low levels of haptoglobin are found in patients with hemolytic anemias. It evaluates the degree of INTRAVASCULAR HEMOLYSIS. Half-life of haptoglobin is approximately 5 days. The half of the Hb-Hp complex is cleared in about 90 minutes, the complex being rapidly removed from plasma by hepatocytes. Haptoglobin-related protein is another protein found in the plasma. Its level is elevated in some patients with cancers. Certain other plasma proteins bind heme but not hemoglobin. Hemopexin is a β1-globulin that binds free heme. Metheme (ferric heme) forms methemalbumin; which transfers the metheme to hemopexin.
  44. 44. β-GLOBULIN β-LIPOPROTEIN: Already discussed in lipid metabolism • Haemopexin: level is very low at birth but reaches adult value by first year of life. normal value: 0.5 – 1.0 g/lit bind and remove circulating haem which is formed in the body from breakdown of Hb, myoglobin or catalase. decrease in hemolytic disorders. •
  45. 45. TRANSFERRIN Transferrin (Tf) is a β1-globulin with a molecular mass of approximately 76 kDa. It is a glycoprotein and is synthesized in the liver. About 20 polymorphic forms of transferrin have been found. Transferrin plays a central role in the body’s metabolism of iron because it transports iron (2 mol of Fe3+ per mole of Tf) in the circulation to sites where iron is required, eg, from the gut to the bone marrow and other organs. Approximately 200 billion red blood cells (about 20 mL) are catabolized per day, releasing about 25 mg of iron into the body.
  46. 46. Transferrin Receptors There are receptors (TfR1 and TfR2) on the surfaces of many cells for transferrin. It binds to these receptors and is internalized by receptor-mediated endocytosis. The acid pH inside the lysosome causes the iron to dissociate from the protein. The dissociated iron leaves the endosome via DMT1 to enter the cytoplasm. Abnormalities of the glycosylation of transferrin occur in the congenital disorders of glycosylation and in chronic alcohol abuse.
  47. 47. Receptor-mediated transferrin endocytosis  Ferro-transferrin binds to the receptors on the cell surface → the complex is internalized into an endosome  In endosomes, iron dissociates from transferrin (enabled by low pH & Fe3+ Fe2+ reduction) and enters cytoplasm  Iron is delivered to intracellular sites or bound to ferritin (Fe2+ Fe3+ oxidation and Fe3+ storage)  Apotransferrin, associated with the receptor, returns to the membrane, dissociates from the receptor and re-enters plasma.
  48. 48. Causes of transferrin deficiency:     Burns Infections Malignancies Liver and kidney diseases Cause of relative transferrin excess:  Iron-deficiency anaemia
  49. 49. C-reactive protein (an acute-phase protein) - It is a protein found in the blood, the levels of which rise in response to inflammation. - Its role is to bind to phosphocholine on the surface of dead or dying cells and some types of bacteria to activate the complement system. - CRP is synthesized by the liver in response to factors released by adipocytes and not related to C-peptide or protein C. • -It is used mainly as a marker of inflammation and in cancer, which is not clear until now. - Measuring CRP values by ELISA, rapid immunodiffusion and visual agglutination is useful in determining disease progress or the effectiveness of treatments.
  50. 50. β-2 MICROGLOBULIN • Present In urine to the extent only 0.01mg/ 100ml • Close resemblance with immunoglobulins. • Increased in renal disease and it is a reflection of impairment of function of glomerular membrane or renal tubules. • Now receiving much attention as a tumour marker. • Useful to check renal damage by gentamycin therapy.
  51. 51. Acute phase reactants (APRs)  Their levels change during acute inflammatory response  APRs concentration changes in:  infection  surgery  injury  cancer
  52. 52. Acute phase reactant response
  53. 53. Types of APRs: Positive 1-antitrypsin C-reactive protein (CRP): ~1000-fold increase! Negative fibrinogen transferrin haptoglobin (HP) C3, C4 albumin
  54. 54. Fibrinogen (0.2-0.45 g/dl) representing 4% of plasma proteins -It is a soluble plasma glycoprotein, that is converted by thrombin into fibrin during blood coagulation. -This is achieved through processes in the coagulation cascade that activate the zymogen prothrombin to the serine protease thrombin, which is responsible for converting fibrinogen into fibrin, which is then cross linked by factor XIII to form a clot. -FXIIIa stabilizes fibrin further by incorporation of the fibrinolysis inhibitors alpha-2-antiplasmin and binding to several adhesive proteins of various cell
  55. 55. α-2 MACROGLOBULIN 2-Macroglobulin is a large plasma glycoprotein (720 kDa) made up of 4 identical subunits of 180kDa. Approximately 10% of the zinc in plasma is transported by 2-macroglobulin , the remainder being transported by albumin.
  56. 56. The protein is synthesized by a variety of cell types, including monocytes, hepatocytes and astrocytes. It is the major member of a group of plasma proteins that include complement proteins C3 and C4. a These proteins contain unique internal cyclic thiol ester bond (formed between cysteine and glutamine residue) and for this reason have been designated as the thiol ester plasma protein family.
  57. 57. 2-macroglobulin binds many proteinases and is thus an important panproteinase inhibitor. The 2-macroglobulin – proteinase complexes are rapidly cleared from the plasma by a receptor located on many cell types. 2-macroglobulinbinds many cytokines (platelet derived growth factor, transforming growth factor β, etc..) and appears to be involved in targeting them toward particular tissues or cells. Once taken up by cells, the cytokines can dissociate from and 2-macroglobulin subsequently exert a variety of effects on cell growth and function.
  58. 58. GENETIC DEFICIENCIES OF PLASMA PROTEINS ANALBUMINAEMIA:  Inherited disorder; very low or complete absence of Albumin.  Defect is in the albumin synthesis  a/w raised lipid and lipoprotein level.  All globulin fraction increased. BISALBUNEMIA:  Two albumin peaks  No clinical importance
  59. 59. BRUTON’S AGAMMAGLOBULINEMIA:  X-linked recessive disorder  Differentiation of B-lymphocytes to plasma cells is defective leading to lack of plasma cells in circulating blood.  Absence of γ-globulin or very low level.  Lack of humoral immunity and susceptible to bacterial infections. AFIBRINOGENEMIA:  Genetic defect in fibrinogen formation.  Absence of fibrinogen or very low level.  Blood clotting mechanism is hampered.  May be uncontrollable hemorrhage.
  61. 61. THANK YOU