BCM 215: Protein in Blood and Structural Protein PDF

BCM 215 PROTEIN IN BLOOD AND STRUCTURAL PROTEIN BY ADEWOLE M.A OBJECTIVES To describe haemoglobin and myoglobin functions To explain physical and chemical properties of haemoglobin and myoglobin To explain how structure of haemoglobin is related to its function...

BCM 215 PROTEIN IN BLOOD AND STRUCTURAL PROTEIN BY ADEWOLE M.A OBJECTIVES To describe haemoglobin and myoglobin functions To explain physical and chemical properties of haemoglobin and myoglobin To explain how structure of haemoglobin is related to its function To elucidate the factors that affect haemoglobin equilibrium To explain Bohr effect using oxygen dissociation curve(ODC) To define haemoglobinopathies and give examples To describe the structure, function and synthesis of collagen To state and explain the abnormalities in collagen To explain the structure and function of elastin Haemoglobin Hemoglobin was one of the first proteins to have its molecular mass accurately determined, the first protein to be characterized by ultracentrifugation, the first to be associated with a specific physiological function (that of oxygen transport), and, in sickle-cell anemia, the first in which a point mutation was demonstrated to cause a single amino acid change. Hemoglobin is not just a simple oxygen tank. Rather, it is a sophisticated oxygen delivery system that provides the proper amount of oxygen to the tissues under a wide variety of circumstances.  Hemoglobin transports oxygen from the lungs, gills, or skin of an animal to its capillaries for use in respiration. Very small organisms do not require such a protein because their respiratory needs are satisfied by the simple passive diffusion of O2 through their bodies. However, since the transport rate of a diffusing substance varies inversely with the square of the distance it must diffuse, the O2 diffusion rate through tissue thicker than 1 mm is too slow to support life. The evolution of organisms as large and complex as annelids (e.g., earthworms) therefore required the development of circulatory systems that actively transport O2 and nutrients to the tissues. The blood of such organisms must contain an oxygen transporter such as Hb because the solubility of O2 in blood plasma (the fluid component of blood).  Although Myoglobin Mb was originally assumed to store oxygen, it is now clear that this function is significant only in aquatic mammals such as seals and whales, which have Mb concentrations in their muscles 10- to 30-fold greater than those in terrestrial mammals. It would seem more likely that Mb’s major physiological role in terrestrial mammals is to facilitate oxygen transport in rapidly respiring muscle. The rate at which O2 can diffuse from the capillaries to the tissues, and thus the level of respiration, is limited by oxygen’s low solubility in aqueous solution.  Mb increases the effective solubility of O2 in muscle, the most rapidly respiring tissue under conditions of high exertion. Hence, in rapidly respiring muscle, Mb functions as a kind of molecular bucket brigade to facilitate O2 diffusion. Whereas hemoglobin is the oxygen-carrying protein of blood, myoglobin is the oxygen-carrying protein of the muscle. Myoglobin is particularly abundant in the muscles of diving mammals, like seals and whales, allowing them to continue to use oxygen even when they are underwater for extended periods of time. Additional physiological functions for Mb have recently been recognized: the detoxification of the highly reactive biological signaling molecule nitric oxide (NO) through its conversion to nitrate ion (NO_3 ) under normal conditions and its synthesis from nitrite ion (NO_2 ) under hypoxic (having an inadequate supply of O2) conditions. Physical and Chemical properties of Haemoglobin and Myoglobin Myoglobin and each of the four subunits of hemoglobin noncovalently bind a single heme group. This is the same group that occurs in the cytochromes and in certain redox enzymes such as catalase. Heme is responsible for the characteristic red color of blood and is the site at which each globin monomer binds one molecule of O2 (globins are the heme-free proteins of Hb and Mb). The heterocyclic ring system of heme is a porphyrin derivative; it consist of four pyrrole rings (labeled A–D in Fig. 1) linked by methene bridges. The porphyrin in heme, with its particular arrangement of four methyl, two propionate, and two vinyl substituents, is known as protoporphyrin IX. Heme, then, is protoporphyrn IX with a centrally bound iron atom. In Hb and Mb, the iron atom normally remains in the Fe (II) (ferrous) oxidation state whether or not the heme is oxygenated (binds O2). The Fe atom in deoxygenated Hb and Mb is 5-coordinated by a square pyramid of N atoms: four from the porphyrin and one from a His side chain of the protein. On oxygenation, the O2 binds to the Fe(II) on the opposite side of the porphyrin ring from the His ligand, so that the Fe(II) is octahedrally coordinated; that is, the ligands occupy the six corners of an octahedron centered on the Fe atom (Fig.1).  Certain small molecules, such as CO, NO, CN, and H2S, coordinate to the sixth liganding position of the Fe(II) in Hb and Mb with much greater affinity than does O2. This, together with their similar binding to the hemes of cytochromes, accounts for the highly toxic properties of these substances. The Fe(II) of Hb and Mb can be oxidized to Fe(III) to form methemoglobin (metHb) and metmyoglobin (metMb). MetHb and metMb do not bind O2; their Fe(III) is already octahedrally coordinated with an H2O molecule in the sixth liganding position. The brown color of dried blood and old meat is that of metHb and metMb. Erythrocytes contain the enzyme methemoglobin reductase, which converts the small amount of metHb that spontaneously forms back to the Fe(II) form. Myoglobin Both Detoxifies and Synthesizes Nitric Oxide NO, which is synthesized in many tissues, functions as a locally active signaling molecule, most notably to induce vasodilation. Once NO has delivered its message, it is important that it be rapidly eliminated to prevent its interference with subsequent NO signals (or lack of them). Moreover, NO is a highly reactive and hence toxic substance. In muscle, under normal O2 concentrations, NO is detoxified through its reaction with oxygenated myoglobin (oxyMb) to yield nitrate ion and metmyoglobin: Since the metMb is subsequently reduced to Mb through the action of an intracellular metmyoglobin reductase, myoglobin functions as an enzyme in this process. Oxygenated hemoglobin (oxyHb) likewise detoxifies the NO that is present in blood. Attachment of Heme with Globin Chain i. There are 4 heme residues per Hb molecule, one for each subunit in Hb. The 4 heme groups account for about 4% of the whole mass of Hb. The heme is located in a hydrophobic cleft of globin chain. ii. The iron atom of heme occupies the central position of the porphyrin ring. The reduced state is called ferrous (Fe++) and the oxidized state is ferric (Fe+++). The ferrous iron has 6 valencies and ferric has 5 valencies. In hemoglobin, iron remains in the ferrous state iii. Iron carries oxygen: The iron is linked to the pyrrole nitrogen by 4 coordinate valency bonds and a fifth one to the imidazole nitrogen of the proximal histidine. In oxy-Hb, the 6th valency of iron binds the O2. The oxygen atom directly binds to Fe, and forms a hydrogen bond with an imidazole nitrogen of the distal histidine. In deoxy-Hb, a water molecule is present between the iron and distal histidine As the porphyrin molecule is in resonance, central iron atom is linked by coordinate bond. The distal histidine lies on the side of the heme ring.  Figure 1: The heme group. Fe(II)–heme (ferroprotoporphyrin IX) is shown liganded to His and O2 as it is in oxygenated myoglobin and oxygenated hemoglobin. Oxygen Binding of Hemoglobin vs. Myoglobin Oxygen diffuses from the alveoli of the lungs- little sacs at the end of the finely divided air passageways in the lung- into the capillaries of the bloodstream and then into the red blood cells, where it binds to hemoglobin. The concentration of oxygen is relatively high in the alveoli, about 100 mm Hg1\. Taking a look at Figure 2, we see that hemoglobin is virtually 100% saturated in the lungs, meaning that essentially all four heme groups have an O2 molecule bound to them. As hemoglobin circulates in the bloodstream to the working muscles, the pressure of oxygen decreases to about 25 mm Hg. At these lower levels of oxygen, hemoglobin is only about 50% saturated. Where did this oxygen go? It was released into the muscles, where myoglobin is found.  Returning to Figure 2, we can see that at 25mm Hg, myoglobin is almost fully saturated, meaning that it will bind the oxygen released by the hemoglobin. The amount of oxygen in the mitochondria, where fuels are burned to release energy, is even lower (1 or 2 mm Hg), allowing myoglobin to release most of its oxygen where it is most needed in the cell. Thus, hemoglobin picks up oxygen in the lungs, circulates through the bloodstream to the muscles (and other tissues), and drops off oxygen there. Myoglobin picks up the oxygen and delivers it to the mitochondria, where it is used to oxidize fuel molecules. The shape of hemoglobin’s oxygen binding curve is sigmoidal (“S”-shaped), with the steep part of the curve occurring at about the oxygen pressure found within the tissues, allowing hemoglobin to deliver a significant amount of oxygen over a fairly narrow range of pressures. That is, it binds oxygen at the relatively high partial pressures in the lungs (the red region in Figure 2) and releases oxygen at the lower partial pressures in the peripheral tissues (the blue region in Figure 2). On the other hand, the shape of myoglobin’s oxygen binding curve is hyperbolic, meaning that it holds onto oxygen much tighter. Only when the amount of oxygen is extremely low, as in the mitochondria of working muscle, does myoglobin release its oxygen. Therefore, the distinct binding curves of these two proteins reflect their functions: hemoglobin, which is well suited for oxygen binding in the lungs, transport in the bloodstream, and delivery to the tissues, and myoglobin, which is well suited for oxygen storage in the muscles and delivery to mitochondria when needed. Figure 2: At higher concentrations of O2, both hemoglobin and myoglobin have more oxygen bound. Hemoglobin’s Structure Influences O2 Delivery The secret to hemoglobin’s success as an oxygen delivery molecule is the fact that it has four subunits that “talk” to each other. Evidence for this is provided by hemoglobin’s “cooperativity” in oxygen binding. In other words, the binding of one O2 molecule affects the binding of others, as we can see by the following:  In order to achieve 25% saturation (an average of 1 O2 molecule per hemoglobin), the amount of O2 needs to be about 18 mm Hg.  In order to achieve 50% saturation (an average of 2 O2 molecules per hemoglobin), the amount of O2 needs to be about 26 mm Hg. Therefore, it is easier to bind the second molecule of O2 than the first. (Otherwise, it would require 2 x 18 mm Hg, or 36 mm Hg, to bind the second.) In order to understand how this is possible, we need to take a detailed look at the structure of hemoglobin. Max Perutz, the scientist who originally determined the structure of the hemoglobin molecule, using a technique called X-ray diffraction, noted during his experiments that hemoglobin can be found in two different forms, or shapes. These different shapes depended on whether oxygen was present or absent, so he called the forms oxy-hemoglobin and deoxy-hemoglobin, respectively. Further experiments revealed that deoxy- hemoglobin has a relatively low attraction for oxygen, but when one molecule of oxygen binds to a heme group, the structure changes to the oxygenated form, which has a greater attraction for oxygen. Therefore, the second molecule of O2 binds more easily, and the third and fourth even more easily. The oxygen affinity of oxy-hemoglobin is many times greater than that of deoxy-hemoglobin. The relationship between these forms can be written as follows: deoxy- hemoglobin + O2 oxy-hemoglobin  The two forms of hemoglobin are said to be in equilibrium with one another because oxygen binding is reversible. Under certain conditions, the deoxy form is favored, and under other conditions the oxy form is favored. Changing the conditions can shift the equilibrium in either direction. For example, adding more O2 would shift the reaction to the right, producing more oxy- hemoglobin. In contrast to hemoglobin, there is only one form of myoglobin. That is, the structure of myoglobin is the same whether oxygen is present or not. The origin of the two different forms of hemoglobin, which account for its cooperative oxygen binding, is the fact that hemoglobin has four subunits. Myoglobin, with its single chain, does not exhibit cooperative oxygen binding. In summary, when O2 binds to a subunit of deoxyhemoglobin, it causes subtle changes in the structure of the protein, altering the way that the four subunits fit together. This in turn affects the structure of each subunit, making it easier for a subsequent molecule of oxygen to bind to the next subunit. Thus, with the binding of the first oxygen molecule to one subunit, the remaining subunits become more receptive to oxygen. Factors that affects Hemoglobin’s Equilibrium Other substances can also alter the binding of oxygen to hemoglobin. In general, such molecules are called “allosteric effectors”. (Greek: allos, other _ stereos, solid or space).These cooperative interactions occur when the binding of one ligand at a specific site is influenced by the binding of another ligand, known as an effector or modulator, at a different (allosteric) site on the protein. If the ligands are identical, this is known as a homotropic effect, whereas if they are different, it is described as a heterotropic effect. These effects are termed positive or negative depending on whether the effector increases or decreases the protein’s ligand-binding affinity. Hemoglobin, as we have seen, exhibits both homotropic and heterotropic effects. The binding of O2 to Hb results in a positive homotropic effect since it increases hemoglobin’s O2 affinity. In contrast, BPG, CO2, H+, and Cl- are negative heterotropic effectors of O2 binding to Hb because they decrease its affinity for O2 (negative) and are chemically different from O2 (heterotropic). The O2 affinity of Hb, as we have seen, depends on its quaternary structure. In general, allosteric effects result from interactions among subunits of oligomeric proteins. Hydrogen ions (protons), CO2, and the molecule 2,3-bisphosphoglycerate (BPG) all promote the release of oxygen by shifting the equilibrium towards the deoxygenated form of hemoglobin. Since these effectors bind to distinct sites, their effects are cumulative. Factors that affect haemoglobin equilibrium Oxygen binding 2,3-biphosphoglycerate (BPG) CO2 H+ and Cl- Oxygen Dissociation Curve (ODC) The strength by which oxygen binds to hemoglobin is affected by several factors and can be represented as a shift to the left or right in the oxygen dissociation curve. A rightward shift of the curve indicates that haemoglobin has a decreased affinity of oxygen, thus, oxygen actively unloads. Figure 4: The presence of acids leads to increased H+ and a reduction in pH. This promotes formation of the deoxy form of hemoglobin, shifting the O2 binding curve to the right. Bohr effect Hydrogen ions and Carbon dioxide are found in high concentrations around actively metabolizing tissues. In the capillaries, binding of these allosteric effectors prompts the release of oxygen from hemoglobin, which can then be taken up by the high affinity myoglobin in the tissues and delivered to the mitochondria. The specific reaction of hydrogen ions and carbon dioxide with hemoglobin causing the release of Oxygen is called the Bohr effect. When deoxygenated hemoglobin returns to the lungs, the concentrations of H+ and CO2 are low. This causes these compounds to be released from hemoglobin. The carbon dioxide is expelled out of the body through expired air. Therefore, hemoglobin not only carries oxygen to the cells, but it also carries  Effect of pH and pCO2 i. When the pCO2 is elevated, the H+ concentration increases and pH falls. In the tissues, the pCO2 is high and pH is low due to the formation of metabolic acids like lactate. Then the affinity of hemoglobin for O 2 is decreased (the ODC is shifted to the right) and so, more O 2 is released to the tissues (Fig. 4). Binding of CO2 forces the release of O2. When the pCO2 is high, CO2 diffuses into the red blood cells. The carbonic anhydrase in the red cells favors the formation of carbonic acid (H 2CO3). Carbonic anhydrase CO2 + H2O ----------------- H2CO3 →H+ + HCO3 When carbonic acid ionizes, the intracellular pH falls. The affinity of Hb for O2 is decreased and O2 is unloaded to the tissues. ii. In the lungs, the opposite reaction is found, where the pCO2 is low, pH is high and pO2 is significantly elevated. More O2 binds to hemoglobin and the ODC is shifted to the left. The Chloride Shift i. When CO2 is taken up, the HCO3¯ concentration within the cell increases. This would diffuse out into the plasma. Simultaneously, chloride ions from the plasma would enter in the cell to establish electrical neutrality. This is called chloride shift or Hamburger effect (Fig. 5). Thus on venous side, RBCs are slightly bulged due to the higher chloride ion concentration. ii. When the blood reaches the lungs, the reverse reaction takes place. The deoxyhemoglobin liberates protons. These would combine with HCO3 – to form H2CO3 which is dissociated to CO2 and H2O by the carbonic anhydrase. The CO2 is expelled. As HCO3 – binds H+, more HCO3– from plasma enters the cell and Cl– gets out (reversal of chloride shift) (See Figure 6) Figure 5:Chloride shift reactions in tissues Figure 6: Chloride shift reactions in lungs  Effect of Temperature The term p50 means, the pO2 at which Hb is half saturated (50%) with O 2. The p50 of normal Hb at 37oC is 26 mm Hg. Elevation of temperature from 20 to 37oC causes 88% increase in p50. Metabolic demand is low when there is relative hypothermia. Shift in ODC to left at low temperature results in release of less O2 to the tissues. On the other hand, under febrile conditions, the increased needs of O2 are met by a shift in ODC to right Effect of 2,3-BPG The 2,3-BPG is produced from 1,3-BPG, an intermediate of glycolytic pathway. The 2,3-BPG, preferentially binds to deoxy-Hb and stabilizes the T conformation. It interacts with deoxygenated hemoglobin beta subunits and decreases the affinity for oxygen and allosterically promotes the release of remaining oxygen molecules bound to the haemoglobin. When the T form reverts to the R conformation, the 2,3- BPG is ejected. During oxygenation, BPG is released. The high oxygen affinity of fetal blood (HbF) is due to the inability of gamma chains to bind 2,3-BPG. Haemoglobinopathies The hemoglobinopathies encompasses all genetic diseases of haemoglobin, there are two main groups which are haemoglobin variants caused by mutations in the haemoglobin gene, and thalassemias which are caused by an underproduction of otherwise normal haemoglobin molecules. Hemoglobin variants Hemoglobin S Hemoglobin E Hemoglobin C Hemoglobin D Hemoglobin M Hemoglobin variants 1. Hemoglobin S (HbS) (Sickle Cell Hemoglobin) Hemoglobin variants, HbS constitutes the most common variety worldwide. 1-A. Sickle Cell Disease i. The glutamic acid in the 6th position of beta chain of HbA is changed to valine in HbS. This single amino acid substitution leads to polymerization of hemoglobin molecules inside RBCs. This causes a distortion of cell into sickle shape (Fig. 7). Fig. 7. Left side—normal RBCs Right side—sickle cells  The substitution of hydrophilic glutamic acid by hydrophobic valine causes a localized stickiness on the surface of the molecule. The deoxygenated HbS may be depicted with a protrusion on one side and cavity on the other side, so that many molecules can adhere and polymerize. HbS can bind and transport oxygen. The sickling occurs under deoxygenated state. The sickled cells form small plugs in capillaries. Occlusion of major vessels can lead to infarction in organs like spleen. Sickle Cell Trait In heterozygous (AS) condition, 50% of Hb in the RBC is normal. Therefore, the sickle cell trait as such does not produce clinical symptoms. Such persons can have a normal lifespan. At higher altitudes, hypoxia may cause manifestation of the disease. Chronic lung disorders may also produce hypoxia-induced sickling in HbS trait. In the electrophoresis, the abnormal HbS can be detected along with normal Hb in persons with HbS trait. HbS gives protection against malaria: The high incidence of the sickle cell gene in population coincides with the area endemic for malaria. HbS affords protection against Plasmodium falciparum infection (Fig. 8). Hence the abnormal gene was found to offer a biologic advantage. Fig. 8: Sickle cell trait protects from malaria Table 1: Important haemoglobinopathies  2.Hemoglobin E It is the second most prevalent hemoglobin variant. It is due to the replacement of beta 26 glutamic acid by Lysine (Table 1). It is primarily seen in orientals of South-East Asia (Thailand, Myanmar, Bangladesh, etc). The variant is very prevalent in West Bengal in India. Heterozygotes are completely asymptomatic. HbE has similar mobility as of A2 on electrophoresis. 3. Hemoglobin C In normals, the 6th amino acid in beta chain is glutamic acid; it is replaced by lysine in HbC (Table 1). The presence of HbC is seen mostly in the black race. AC heterozygotes do not show any clinical manifestations. But those who are double heterozygous for HbS and HbC (SC) have a moderate disease. Homozygotes (CC) have a mild to moderate hemolytic anemia. The HbC is slower moving than HbA on electrophoresis at alkaline pH. 4. Hemoglobin D It does not produce sickling. HbD Punjab results from replacement of beta 121 glutamic acid by glutamine (HbD) 5. M-Hemoglobins (Hb M) These are a group of variants, where the substitution occurs in the proximal or distal histidine residues of alpha or beta chains. Alpha 58 His →Tyr (Hb M Boston) Beta 92 His →Tyr (Hb M Hyde Park) Thalassemia Thalassemia may be defined as the normal globin chains in abnormal proportions. The gene function is abnormal, but there is no abnormality in the polypeptide chains. Reduction in alpha chain synthesis is called alpha thalassemia, while deficient beta chain synthesis is the beta thalassemia. STRUCTURAL PROTEINS: COLLAGEN AND ELASTIN The major structural protein found in connective tissue is the collagen. Collagen is a Greek word which means the substance to produce glue. It is the most abundant protein in the body. About 25-30% of the total weight of protein in the body is collagen. It serves to hold together the cells in the tissues. It is the major fibrous element of tissues like bone, teeth, tendons, cartilage and blood vessels. Fig. 9: Triple stranded collagen fiber Table 2: Post-translational processing of collagen Abnormalities in collagen 1. Osteogenesis Imperfecta 2. Ehlers-Danlos Syndrome (EDS) 3. Alport Syndrome 4. Epidermolysis bullosa 5. Marfan's Syndrome 6. Menke's Disease 7. Deficiency of Ascorbic Acid 8. Homocystinuria Elastin Elastin is a protein found in connective tissue and is the major component of elastic fibers. The elastic fibers can stretch and then resume their original length. They have high tensile strength. Disease associated with Abnormalities in Elastin Williams-Beuren syndrome Pseudoxanthoma elasticum Copper deficiency