2.2025. MPF Simple proteins and Hemoglobin.pptx

Classification of simple proteins. Hemoglobin properties. Bohr effect, cooperativeness, rT-and R-forms

1. This Photo by Unknown Author is licensed under CC BY In The Name of Allah, The Most Merciful, The Most Compassionate

2. Simple and complex proteins. Hemoglobin, its hetero­geneity and cooperativeness. Immunoglobulins. Dr. Mahira Amirova Associate Professor Biochemistry Department of Azerbaijan Medical University Year 2025, fall term

3. Simple proteins are sort due to solubility to: 1) Scleroproteins (not dissolved in water and saline solutions) 2) Albumins and Globulins (differently dissolved in water and saline solutions) 3) Prolamins and glutelins (dissolved in 70% alcohol solution) 4) Protamines and histones (dissolved in acidic medium)

4. Scleroproteins are the m ajor proteins of connective tissue: collagen and elastin, that are synthesized in fibroblasts Unlike most globular proteins that are folded into compact structures, a fibrous protein collagen , has an elongated triple-helix form.

5. In general, Scleroptorteins are Fibrous Proteins. Collagen, Elastin, Keratin are referred to them. Collagen is a fibrous protein synthesized as pre-pro-collagen, is converted in extracellular space to tropocollagen. Collagen has an elongated, triple-helical structure, where 3 polypeptide α chains are held together by interchain hydrogen bonds. Collagen is rich in proline and glycine, both of which are important in the formation of the triple-stranded helix.

6. Unusual amino acids found in Collage n Glycine is found in every third position of the polypeptide chain. It fits into the restricted spaces where the three chains of the helix come together. Glycine is part of a repeating sequence, –Gly–X–Y–, where X is frequently proline, and Y – hydroxyproline, Hyp (or hydroxylysine, Hyl ). Thus, α chain can be represented as (–Gly–Pro– Hyp –)333

7. Collagen chain sequence

8. Hydroxyproline ( Hyp ) – for interchain hydrogen bonds and hydroxylysine ( Hyl ) – for glycosylation Collagen contains hydroxyproline and hydroxylysine, which are not present in most other proteins. These residues result from the hydroxylation of some of the proline and lysine residues after chain synthesis on ribosome. Hydroxyproline maximizes formation of interchain hydrogen bonds that stabilize the triple-helical structure. Glucose and galactose are sequentially attached to hydroxylysine in the polypeptide chain prior to triple-helix formation

9. Collagen structure

10. Collagen synthesis

11. Maturation of Collagen

12. Intracellular and extracellular phases

13. In extracellular space : Procollagen -> Tropocollagen

14. Peptidases (N- proteinase and C-proteinase) action on procollagen triplet > tropocollagen

15. Lysyl oxidase action on tropocollagen and collagen self assembly

16. Cross-linking at Lys and Hyl

17. Ehleros-Danlos disease

18. https://www.bonebrothstock.com/wp-content/uploads/2017/06/diet.png During healing – collagen rich diet Hoof broth is preferable. Such diet is absolutely indispensable during recovery after surgery to reconnect damaged tissues.

19. Elastin. Desmosine and isodesmosine . Four lysins associate into unit of elastin, namely desmosine Desmosine is a constituent of 4 elastin molecules simultaneously, making linkage between these 4 chains.

20. Desmosine connects with molecules of collagen, hyaluronic acid, adhesive proteins Desmosine linked with different compounds collagen H yaluronic acid tanassin

21. Elastin is rich in hydrophobic amino acids. Elastin elasticity : EXTENDED and RELAX forms. the stretching force is relaxed U nder high tension

22. Keratin ( scleroprotein ) in hair When exposed to mechanical stress,  α - keratin  structures can retain their shape and therefore can protect what they surround.

23. Under high tension,  alpha - keratin  can even change into  beta - keratin After heating, α -keratin turns to β -keratin α -keratin the wool sweater will shrink in hot water, since α -keratin turns to more compact and stable β -keratin

24. KERATIN IS RICH IN CYSTEINE

25. C URLING OF HAIR is based on cystein reducing-oxidation properties curling of hair : cysteine oxidation scheme

26. Simple globular proteins. ALBUMINS AND GLOBULINS

27. Albumin characteristics

28. Proteins total concentration in the blood (total blood protein = 65-85g/l) 40-50

29. Globulin fractions are: α 1 -, α 2 -, β 1 -, β 2 -, γ- globulins . pseudo globulins precipitated with 33% solution of ammonium sulphate Euglobulins , that precipitate in 50% solution of ammonium sulphate

30. Each blood fraction comprises the clinically important proteins of Each blood fraction comprises clinically important proteins In blood plasma,  haptoglobin  binds to free hemoglobin, compared to  hemopexin  that binds to free heme released from erythrocytes with high affinity, and thereby inhibits its deleterious oxidative activity. The  haptoglobin -hemoglobin complex will then be removed by the reticuloendothelial system (mostly the spleen).

31. Gamma Globulins: Components: Immunoglobulins (IgG, IgA, IgM, IgD , IgE ). Function: Antibody-mediated immune response. Clinical Significance: Elevated levels may indicate chronic infections, autoimmune disorders, or plasma cell disorders. Gamma-globulins in h uman Blood Protein Fraction (Proteinogram)

32. Blood albumins and globulins: separation of blood proteins by electrophoresis Blood albumin does not contain glycocol, its isoelectric point = 4.7, just as isoelectric point of casein (present in milk) and ovalbumines. The ratio of albumin to globulins in the blood is referred to as serum protein index , in the norm it is = 1.5 -2.3 . Milk contains lactalbumin ; ovalbumin is an egg protein.   (confront antigen)

33. ALBUMINS AND GLOBULINS of blood plasma can be represented this way:

34. A. Norm B. Infectious disease Schematic

35. PROTEINOGRAM (changes in protein ratio in different circumstances, diseases) – real electrophoresis results

36. Understanding the proteinogram and its changes in pathology is crucial for diagnosing and monitoring various diseases. It provides valuable information about the underlying physiological processes, organ function, and the body's response to stressors. Interpretation often involves considering the entire protein profile rather than isolated changes in individual fractions. Proteinogram c hanged in Pathology:

37. Liver Disorders: Decreased synthesis of albumin and other proteins in liver diseases, leading to hypoalbuminemia. Changes in alpha and beta globulins due to impaired hepatic synthesis. Inflammation and Infection: Elevated levels of acute-phase proteins (alpha-1 globulins) in response to inflammation or infection. Changes in gamma globulins may indicate chronic infections or immune system activation. Changes in the Proteinogram in Pathology:

38. 3. Kidney Diseases: Proteinuria, the presence of excess proteins in urine, can lead to a decrease in albumin levels in the blood. Altered alpha and beta globulins in some renal conditions. 4. Malnutrition: Decreased levels of albumin due to malnutrition or protein-losing conditions. Changes in protein fractions reflect nutritional status. Changes in the Proteinogram in Pathology:

39. 5. Hematological Disorders: Elevated gamma globulins in plasma cell disorders like multiple myeloma . Changes in beta globulins in conditions like hemoglobinopathies. 6. Autoimmune Diseases: Altered gamma globulin levels may indicate autoimmune diseases where antibodies attack the body's own tissues. Changes in the Proteinogram in Pathology:

40. PROLAMINS AND GLUTELINS

41. What is gluten? - a protein of grain Coconut:

42. https://i.pinimg.com/originals/28/7f/cc/287fcce6145a54bf2e3b414307e8572e.jpg Gluten from different grains

43. Celiac disease Gluten – glutelin + prolamin N ormal villi atrophic villi bloating, flatulence in the intestine

44. Gluten present in most of delicacy: Gluten is the general term for a protein found in  wheat ,  barley ,  rye , and so on. All forms of  wheat  contain gluten. Many everyday food products have gluten, such as  pasta ,  bread. Beer also comprises gluten.

45. Complex proteins

46. HEMEPROTEINS: MYOGLOBIN, HEMOGLOBIN, CATALASE, CYP 450… Myoglobin of muscle: 1 CHAIN + 1 heme with SINGLE IRON IN IT All contain heme (iron with protoporphyrine IX in center)

47. Structure and function of myoglobin Myoglobin, a hemeprotein in heart and skeletal muscle, functions both as a reservoir for oxygen. Myoglobin consists of a single polypeptide chain combined with heme Myoglobin can bind only one molecule of O 2 , because it contains only one heme group Myoglobin is designed to bind oxygen released by hemoglobin at the low pO2 found in muscle. Myoglobin, in turn, releases oxygen within the muscle cell in response to oxygen demand.

48. M y oglobin vs. Hemoglo b i n Mb is monomer, Hb is a tetramer (ex. a2b2). Hb subunits are structurally similar to Mb, with 8 a-helical regions, no b-strands and no interior water. Both contain one heme prosthetic group per chain. Both Mb and Hb contain proximal and distal histidines. Affinity of Mb for oxygen is high, affinity of Hb for oxygen is lower and more variable .

49. Structure of heme Heme is a complex of protoporphyrin IX and ferrous iron (Fe2+). The iron in the center heme has 6 bonds: 4 are used to linkage with 4 nitrogens of porphyrin ring. The heme Fe 2+ can form two additional bonds. In myoglobin and hemoglobin, one of these positions is coordinated to histidine of globin, whereas the other position is available to bind oxygen or any other ligand.

50. Heme= iron+ protoporphyrin IX heme

51. Model of myoglobin showing myoglobin (Mb). B. Schematic diagram of the oxygen-binding site of myoglobin.

52. Structure and function of hemoglobin Hemoglobin is found in red blood cells (RBC), where its main function is to transport oxygen (O2) from the lungs to the capillaries of the tissues. Hemoglobin A, the major hemoglobin in adults, is composed of four polypeptide chains ( two α chains and two β chains ) held together by noncovalent interactions. Hemoglobin can transport H + and CO 2 from the tissues to the lungs and can carry 4 molecules of O 2 from the lungs to the cells of the body.

53. Quaternary structure of hemoglobin A1 The hemoglobin tetramer is composed of two identical dimers, (αβ)1 and (αβ)2. The two polypeptide chains within each dimer are held tightly together primarily by hydrophobic interactions. In contrast, the two dimers are held together primarily by polar bonds .

54. In adults, HbA 1 ( 2 is main protein of RBC, as accounts 95-98% of total blood hemoglobin  

55. HEMOGLOBIN properties: HETEROGENEITY Main normal hemoglobin types in adult are: Hemoglobin A (Hb A 1, 2 ), which is 95-98% of hemoglobin found in adults and Hemoglobin A 2 ( Hb A 2: 2 , which is 2-3% of hemoglobin found in adults. Hemoglobin F (Hb F, 2 ), produced in fetus is found in adults up to 2.5%. T here is also synthesized the primary hemoglobin (HbP) by the fetus, called Gover- hemoglobin. , Hb Gover1 consisting of 2 alpha-like is produced in fetus, 2 - and 2  

56. Hb Gower and HbF HbF synthesis during development: In the first month of fetus, embryonic hemoglobins such as Hb Gower 1 , composed of two α-like zeta (ζ) chains and two β-like epsilon (ε) chains ( ζ2ε2 ), are synthesized by the embryonic yolk sac . In the fifth week of gestation, the site of globin synthesis shifts, first to the liver and then to the marrow, and the primary product is HbF .

57. HbF HbF is the major hemoglobin found in the fetus and newborn, HbA synthesis starts in the bone marrow at about the eighth month of pregnancy and gradually replaces HbF . HbF represents less than 1% of the hemoglobin in most adults

58. Hemoglobin A2 HbA2 is a minor component of normal adult hemoglobin, first appearing shortly before birth and, ultimately, constituting about 2% of the total hemoglobin. It is composed of two α-globin chains and two δ-globin chains ( α2δ2 )

59. https://www.facebook.com/photo.php?fbid=590144544794616&id=229353567540384&set=a.229376704204737

60. Loading and unloading of O 2 by Hemoglobin (R- and T-forms) T form is the deoxy form of hemoglobin called “T, ” or taut (tense) form. The T conformation is the low-oxygen affinity form of hemoglobin. This form is prevalet in the periferic tissues. R form is the high-oxygen-affinity form of hemoglobin. The binding of O 2 to hemoglobin causes the rupture of some bonds between the αβ dimers leading to formation of “R, ” or relaxed structure. This form is prevalet in the lungs.

61. OxyHb (R) coming from the lungs, is converted to deoxyHb (T) in the tissues

62. Cooperativeness The sigmoidal oxygen-dissociation curve reflects that the affinity of hemoglobin for the last oxygen bound is approximately 300 times greater than its affinity for the first oxygen bound. Reference for picture: https://ditki.com/course/biochemistry/glossary/biochemical-pathway/hemoglobin-myoglobin-4-dissociation-curves

63. Oxygen-dissociation curve A plot of saturation measured at different partial pressures of oxygen (pO2) is called the oxygen-dissociation curve. The curves for myoglobin and hemoglobin show important differences. Myoglobin has a higher oxygen affinity at all pO2 values than does hemoglobin. The partial pressure of oxygen needed to achieve half-saturation of the binding sites ( P 50 ) is approximately 1 mm Hg for myoglobin and 26 mm Hg for hemoglobin .

64. If hemoglobin can’t absorb the excess protons, H + remains in the blood, & the acidity raises. This increased acidity serves a two fold purpose. In the presence of the high oxygen concentration in the lungs, the proton affinity to Hb is decreas ed . As protons are shed, CO 2 forms as an insoluble gas to be expelled from the lungs. THE GENEROUS BUFFER SYSTEM OF BLOOD is Hb

65. Bohr effect The release of oxygen from hemoglobin is enhanced when the pH is lowered or when the hemoglobin is in the presence of an increased pCO 2 . Both result in a decreased oxygen affinity of hemoglobin This change in oxygen binding is called the Bohr effect. Conversely, raising the pH or lowering the concentration of CO 2 results in a greater affinity for oxygen, a shift to the left in the oxygen-dissociation curve, and stabilization of the R (oxy) form

66.   H+, CO2, BPG Hb T & R forms interconvertion b a b a R (high affinity) O 2     T (low affinity ) The ability of hemoglobin to release oxygen to the tissues is affected by pH, CO 2 and BPG

67. I n the tissues pH is low, in the lungs - high Bohr effect: the release of oxygen from hemoglobin is enhanced when the pH is lowered. Conversely, raising the pH at lowering of the concentration of CO 2 results in a greater affinity of hemoglobin for oxygen, stabilizing the R (oxy) form of hemoglobin.  

68. Released from the tissues protons lower the affinity of hemoglobin for oxygen, allowing easier release into the tissues. As all four oxygens are released, hemoglobin binds to two protons. This helps to maintain equilibrium towards the right side of the equation. This is known as the Bohr effect , and is vital in the removal of carbon dioxide as waste because CO 2 is insoluble in the bloodstream. The bicarbonate ion is much more soluble .

69. https://byjus.com/chemistry/bohr-effect/

70. Allosteric effects: 2,3-bisphosphoglycerate The ability of hemoglobin to bind oxygen is affected by the pO2, pH of the environment, the partial pressure of carbon dioxide (pCO2) and availability of 2,3-bisphosphoglycerate. The binding of oxygen to monomeric myoglobin is not influenced by allosteric effectors.

71. Binding site of 2,3-BPG N egatively charged phosphate groups of 2,3-BPG bind to charged amino acids via ionic bonds in pocket of two β-globin chains in the center of deoxy hemoglobin. The concentration of 2,3-BPG in the RBC increases in response to chronic hypoxia or at high altitudes, chronic anemia. Since levels of 2,3-BPG in RBC decrease during blood storage for transfusion, inositol is added to blood to help restore hemoglobin's ability to release oxygen to tissues

72. BUT 2,3-BPG FACILITATES OXYGEN RELEASE WITH FORMATHION T-Hb 2,3-Bisphosphoglycerate ( 2,3-BPG) is the most abundant organic phosphate in the red blood cells (RBC), where its concentration is approximately that of hemoglobin. 2,3-BPG is synthesized from an intermediate of the glycolytic pathway. The concentration of 2,3-BPG in the RBC increases in response to chronic hypoxia , such as that observed in chronic obstructive pulmonary disease like emphysema, or at high altitudes, where circulating hemoglobin may have difficulty receiving sufficient oxygen.

73. BPG e ffect on Hb saturation 2,3,- B P G helps Hb dissosiation 20 40 60 80 100 120 140 160 pO 2 (mm Hg) 2 4 6 8 1 pO2 vs p50=8 pO2 vs p50=26 Hb alone Hb + BPG 2,3- B PG is the main player in Hb coopera tivity. High altitude increases 2.3- BPG, pushing curve further to right

74. Intracellular levels of 2,3-BPG 9 (in RBC) are also elevated in chronic anemia , in which fewer than normal RBCs are available to supply the body’s oxygen needs. Elevated 2,3-BPG level lowers the oxygen affinity of hemoglobin, permitting greater unloading of oxygen in the capillaries of the tissues. To increase the synthesis of 2,3-Bisphospho - glycerate in transfused blood , inosine is added to it .

75. Binding of CO2 with Hb occurs via N- terminal amino acid Most of the CO 2 produced in metabolism is transported as bicarbonate ion. However, some CO 2 is carried bound to the N-terminal amino groups of hemoglobin (forming Carbhemoglobin ) . The binding of CO 2 stabilizes the T or deoxy form of hemoglobin , resulting in a decrease in its affinity for oxygen . In the lungs, CO 2 dissociates from the hemoglobin and is released in the breath.

76. Binding of CO and NO Carbon monoxide (CO) binds tightly (but reversibly ) to the hemoglobin iron, forming carboxy hemoglobin. As a result, the affected hemoglobin is unable to release oxygen to the tissues . The affinity of hemoglobin for CO is ~300 times greater than for oxygen. Consequently, even minute concentrations of CO in the environment can produce toxic concentrations of carboxyhemoglobin in the blood.

77. Binding of CO İncreased levels of CO are found in the blood of tobacco smokers CO poisoning is treated with 100% oxygen at high pressure (hyperbaric oxygen therapy), which facilitates the dissociation of CO from the hemoglobin. CO also inhibits Complex IV of the electron transport chain . In addition , nitric oxide gas (NO) also is carried by hemoglobin. NO is a potent vasodilator

78. CO connection to Hemoglobin When incomplete combustion of gas methane occurs, CO concentration rises in inhaled air and poisoning with carbon monoxide occur, i.e. formation of tightly bound form of carboxyHb in the blood. CO is found in fumes produced any time we burn fuel in cars, fireplaces, gas ranges . CO can build up indoors and poison people and animals who breathe it. Formation of carboxyHb in the blood leads to death.

79. The most common symptoms of CO poisoning are headache, dizziness, weakness, upset stomach, vomiting, chest pain, and confusion. CO symptoms are often described as “flu-like.” If you breathe in a lot of CO, it can make somebody pass out or kill . People who are sleeping or drunk can die from CO poisoning before they have symptoms.

80. Methemoglobinemia ( MetHb ) Oxidation of the heme iron in hemoglobin to the ferric (Fe3+) state forms methemoglobin ( MetHb ), which cannot bind O 2 . This oxidation may be caused by the action of certain drugs, such as nitrates, Bertholet's salt or endogenous reactive oxygen species (ROS). The methemoglobinemia treatment is with methylene blue, which reduces Fe+3

81. CHAINGE OF IRON VALENCY results in MET-Hb formation

82. Methemoglobinemia in some African indigenous aborigines B. Norm A. Met Hb- emia