Blood Proteins and Their Functions

1. Blood - Biochemical AspectsFunctions • Respiratory • Transport O2 from lungs to tissues • Transport CO2 from tissues to lungs • Nutrition • Transport “food” from gut to tissues (cells) • Excretory • Transport waste from tissues to kidney (urea, uric acid, water)

2. Regulatory • Water Content of Tissues • Water exchanged through vessel walls to tissue (interstitial fluid) • Body Temperature • Water- high heat capacity, thermal conductivity, heat of vaporization • Typical heat generation is 3000 kcal/day • Protective • Antibodies, antitoxins, white blood cells (WBC)

3. Blood composition • 5-6 L in an adult • 70 mL/kg of body weight • Suspension of cells in a carrier fluid (plasma) • Cells - 45% by volume • Plasma - 55% by volume • Cells • Red cells (erythrocytes) • 5x106/mL • White cells (leukocytes) • 7x103/mL • Platelets (thrombocytes) • 3x105/mL

4. Plasma composition • Water - 90% of plasma volume • Proteins - 7% of plasma volume • Inorganic - 1% of plasma volume • Na+, K+, Mg2+, Ca2+, PO43-… • Organic - 2% of plasma volume • urea, fats, cholesterol, glucose ...

5. Male versus female • Hematocrit (% volume that is red cells) • 40-50% in males • 35-45% in females

6. ProteinsSee Lehninger Chapter 3-6 • Proteins are polyamino acids • Macromolecules - MW 5000 - several million • Insulin - MW = 6000 • Hemoglobin - MW = 68 000

7. O O ~NHCHC-NHCHC~ R1 R2 Peptide bond R NH2CH COOH Amino Acid Structure Protein Structure

8. 20 common amino acids (AA) • Classified based on the properties of the R groups Acidic Glutamic Acid Basic Lysine

9. Polar Tyrosine Apolar Glycine

10. Amino Acids and Proteins • Acidic and basic groups are charged at blood / physiologic pH • Proteins are polyelectrolytes • pH of zero net charge (pI or isoelectric point) depends on amino acid composition of protein • Blood proteins negative at pH 7.4 • more COO- than NH3+, pI < 7.4

11. pI • Protein has many negative charges • Requires H+ to neutralize • Therefore low pI • Consider a protein with pI = 4 • If pH increases above pI protein becomes? • If pH decreases below pI protein becomes? • Higher the pI the more +,- is protein?

12. Need to go to a higher pH to neutralize or compensate for + charges • Minimum solubility occurs at pI since there is no intermolecular repulsion • At pH 7.4 (blood pH), all blood proteins are negative and therefore have pI’s less than 7.4

13. Protein Structure • Four levels • Primary structure: sequence of amino acids • 20 amino acids in long chain molecules • many possible combinations • Secondary structure: arrangement of the chains in space (conformation of chains) • a-helix: coil shape (due to H bonding) • b-sheet: stretched zig-zag peptide chain (H bonding • random coil: similar to synthetic polymers

18. Tertiary structure: folding of chains into 3 dimensional shape due to H bonding, S-S bonds and hydrophobic interactions • Several different types of secondary structure within the full three dimensional structure of a large protein • Quaternary structure: present in proteins with several polypeptide chains, arrangement and interelationship of the chains due to S-S bridging • Four levels result in well defined shape and chemical structure essential for function of protein

19. Plasma Proteins • More than 200 • Most abundant • Albumin - 4-5 g/100 mL • g-glubulins - ~1 g/100 mL • fibrinogen - 0.2-0.4g/100 mL • Original classification by zone electrophoresis at pH 8.6 • Separation by pI with several molecular weight species within each group

20. Zone Electrophoresis of Plasma Proteins + - globulins albumin g b a1 a2 pI 6.0 5.6 5.1 4.7

21. Protein Separation • Size Exclusion Chromatography (SEC) • Porous matrix (sephadex)

22. Affinity chromatography • molecule attached to a column that specifically binds the protein of interest • Coenzyme / enzyme • Antigen / Antibody

23. SDS-PAGE (polyacrylamide gel electrophoresis) • Separates by size • Proteins are complexed with SDS to give the same charge density

25. Two Dimensional Electrophoresis Decreasing Mr Decreasing pI

26. Functions of Plasma Proteins • Maintenance of: • Colloid osmotic pressure (p) • pH • electrolyte balance • COP relates to blood volume DP = p Protein sol’n Water

27. If membrane present p important • “Isotonic” - same osmotic pressure • Human blood - 300 milliOsmoles /L • Normal saline - 0.9% NaCl by weight • 0.15 mol/L • 0.30 mol/L of particles • Calculate osmotic pressure from concentration?

28. By analogy with the ideal gas law • In blood, which protein contributes most to p? • Low molecular weight, high concentration

29. Colloid - large particle that cannot easily cross a membrane • Stays in the compartment • In blood pprotein = 20-30 mmHg • Total ~ 5000 mmHg • Protein stays in the blood as p is maintained in the blood • Water content is therefore maintained

30. H2O Hb • Hypotonic - lower p than normal • Hemolysis of RBC H2O Ghost Cells • Hypertonic - higher p than normal • Hemolysis of RBC Crenated Cells Hypertonic 1.5% NaCl

31. Functions of Plasma Proteins (cont’d) • Transport of ions, fatty acids, steroids, hormones etc. • Albumin (fatty acids), ceruloplasmin (Cu2+), transferrin (Fe), lipoproteins (LDL, HDL) • Nutritional source of amino acids for tissues • Hemostasis (coagulation proteins) • Prevention of thrombosis (anticoagulant proteins) • Defense against infection (antibodies, complement proteins)

32. Function and Properties of Selected Plasma Proteins • Consider three abundant plasma proteins • Structure, function • Coagulation, fibrinolysis, complement

33. Albumin • MW 66 000 • Single chain, 580 amino acids, sequence is known • Dimensions - Heart shaped molecule • 50% a helix[He and Carter, Nature, 358 209 (1992)] • Modeled as: 80 Å 30 Å

34. Synthesis • Mainly liver cells then exported • Assembly time on ribosome ~ 1-2 min • t0.5 in circulation - 19 days • 14 g lost per day • 0.4 mg synthesized per hour per g of liver • Need liver of approximately 1.5 kg in weight to maintain

35. Functions • “Colloid” osmotic pressure of blood is 80% due to albumin • relatively low molecular weight • regulates water distribution • Transport of fatty acids • Liver to tissues, binding • Source of amino acids for tissue cells (pinocytosis) • 60% albumin in tissue (interstitial) fluid

36. g-Globulins • 20% of plasma proteins • “g” refers to electrophoretic mobility • Represents a group of proteins of variable structure • immunoglobulins • Main functional task is immunochemical • Antibodies - combine with specific antigens

37. Basic 4 chain structural unit • MW = 2x55000 +2x27000 = 160000

38. Variable region varies with respect to primary, secondary and tertiary structures • Basis of specificity of antigen binding (106 average number) • 5 classes of immunoglobulins • IgG, IgA, IgM, IgD, IgE • Different structures of constant regions of heavy chains • Some are polymers (multiples of 4 chain unit - IgA - dimer - MW 350 000, IgM - pentamer - MW 900 000 • See any immunology book for more details

39. Functions • Primary function is antigen binding (immune response) • Secondary function is complement binding (after antigen) • Each class has different functions • IgE - allergic reactions (defence) • IgA - secretory protein, high concentration in external fluids (saliva, tears) • IgD - ? Involved in differentiation of B lymphocytes (found on the surface of B-lymphocytes)

40. Synthesis • In lymphocytes (T and B) • Made in response to presence of antigen (“foreign” macromolecule, virus particle etc.)

42. Fibrinogen • Coagulation • Structure • MW 340 000 • Sequence of amino acids is known (3000) • 4y, 3y structure • 6 polypeptide chains, 2a (67,000), 2b (56,000), 2g (47,000)

43. a b g disulfide Triple dumbell model (EM) 450 Å 90 Å D E D a’s, b’s and g’s are intertwined

44. Thrombin Fibrinogen Fibrin Plasmin Fibrin Degradation (FDP) • Function • Blood coagulation (clotting) • Plasmin is end product of fibrinolytic system • Clot needs to be removed • Not needed forever • Could embolize to lungs, brain

45. Sickle Cell Anemia • Occurs because of a minor variation in one amino acid in the b chain of Hb • Results in Hb that, when exposed to low O2 concentrations precipitates into long crystals • Elongate cell • Damage cell membrane • Decrease in amount of RBC

46. Cellular Elements of Blood • Red cells • 40 - 50% of blood volume • 5 x 106 cells /mL • “bag” of hemoglobin • non-nucleated • no proliferation • cell membrane in excess so that deformation does not rupture • Shape • Biconcave disc • 8 mm in diameter, 2.7 mm thick, volume ~ 90 mm3, area ~ 160 mm2

47. Scanning Electron Micrograph of Red Blood Cells

48. Why this shape? • Area to volume ratio is high (maximal?) • Facilitates diffusion of O2 and CO2 • minimal distance of contents from surface • Originates in bone marrow (hematopoiesis) • Molecular explanation based on the properties of the proteins in the cell membrane is found in Elgsaeter et al. Science, 234, 1217 (1986)

49. Oxygen Binding of Hb • Blood must carry 600 L of O2 from lungs to tissues each day • Very little carried in plasma since O2 only sparingly soluble • Nearly all bound and transported by Hb of RBC • Possible for Hb to carry four O2 molecules, one on each a chain, one on each b chain

50. O2 depleted Hb solution placed in contact with O2(g) • Equilibrium reaction • Fraction (s) of Hb converted to oxyhemoglobin