Chromoproteins. Pathological and physiological forms of hemoglobin
Hemoglobin is a conjugated protein, consisting of simple protein globin and prosthetic group – hem. Hem is helatic complex of the porphyrin with an iron atom in the center. Porphyrin is cyclic compound, which has 4 pyrrole groups, joined together by methane bridges. There are different porphyrins that differ from each other by lateral groups of the pyrrole rings. Heme forms part of mioglobin, cytochrom-b, cytochrome P-450, catalases, peroxydases too (heme-containing proteins). Ion of Fe is combined with four atoms of nitrogen of pyrrole rings (2 covalent bonds, 2 donor-acceptic bonds) and by coordination bond with atom of nitrogen of histidine imidazole group in polypeptide chain.
Globin is protein consisting of four polypeptide chains (2a chains – each of them contains 141 amino acid residues, 2b chains containing of 146 amino acidic residues). Every chain is connected only with one heme molecule. Four polypeptide subunits in space in the form of tetrahedron in a compact packaged gives us globular molecule, where subunits are connected with each other. This is a main form of hemoglobin of adult person – hemoglobin A. Approximately 2 % of human hemoglobin is hemoglobin A2, which contains d-chains instead of β-chains (2a2d). Fetus has HbF (fetal), consisting of 2a and 2g chains and forming 80 % of Hb. At the last week of pregnancy and the first days after the birth HbF gradually changes to HbA, and after 1st year content of Hb F is near 1,5 %. HbF has greater affinity for oxygen than does HbA that is why fetus can pick up oxygen from the maternal bloodstream.
In human blood nearly 300 variations of Hb are discovered, appeared as a result of gene mutations, but majority of them don’t cause some disease. One of most important anomalous hemoglobin is HbS. People – germ carriers of gene HbS has sickle cell anaemia that is hemolytic by the mechanism of development. HbS differs from HbA by the substitution of the one amino acid at 6th position of b chain, where glutamate is substituted by valine. These amino acids are different by the charge and hydrophobic interactions, the substitution lower the solubility of HbS in deoxyform. Molecules of deoxyhemoglobin form the threads, fibers and bunch of fibers, which cause the change of the erythrocytes form. Sickle cells are less stable than normal ones and are destroying very fast. The blood of homozygotic people has only HbS and the severe anemia are developed, death comes in early childhood. The blood of the heterozygotic people has HbS and HbA, so only the poor symptoms of disease appear. In such individuals the process of development of malarial plasmodium delayed and they do not ill to malaria or can easy cope with it. Gene HbS is common in malaria regions.
The relation of some anomaly hemoglobins to the oxygen increases or decreases which also can cause to hematolytic diseases. Besides these hemoglobins diseases are hereditary ones as a result of dysfunction producing a and b chains in equal quantities or the absolute absence of synthesis of one kind of chain. These diseases are called talassemia. The result of misbalance a and b chains is that superfluous chains sedimantats the level of hemoglobin and the life period of erythrocytes decrease. Homozygotic form of talassemia is resulted to death at the prenatal or neonatal periods.
Oxygen transport is one of the main blood functions. Only minor quantity of the O2 is transported in soluble form, whereas bound with hemoglobin quantity is greater in 70 times. One molecule of the hemoglobin consisting of four hems can bind four molecules of the oxygen. Joining of oxygen doesn’t change the iron valency, because it is providing by coordination bonds of iron molecule. Hemoglobin bound with oxygen is called oxyhemoglobin (oxygenated hemoglobin).
Carbonate is transported by the blood in soluble form - 6-7 %, bound with hemoglobin (carbhemoglobin) – 3-10 %, and as hydrocarbonates – 80 %.
In carbhemoglobin molecule CO2 is connected with N-end of each from 4 polypeptide chain. This compound is very unstable and can dissociate in lungs capillaries with CO2 chipping off.
Carboxyhemoglobin is connection of hemoglobin with carbon monoxide CO. The affinity of hemoglobin to CO is in 200 times higher than to O2. Very small concentration of CO in air has toxic effect on the organism. When one part of gem group is connected with CO and other one with O2, molecules of hemoglobin give oxygen worse than hemoglobin in connection with 4 molecules of oxygen. So in poisoning CO hypoxia is making not only by connection part of hems with CO but also the shift of oxyhemoglobin dissociation.
The Fe2+ in hemoglobin is susceptible to oxidation to Fe3+ by superoxide and other oxidizing agents (amilnitrit, aniline, nitrobensol, nitrates and nitrites, tiosulfats, fericianid), forming metHb, which cannot transport oxygen. Only a very small amount of metHb is present in normal blood, as the RBC possesses an effective system (the NADH-cytochrome b5 methemoglobin reductase system) for reducing heme Fe3+ back to the Fe2+ state. This system consists of NADH (generated by glycolysis), a flavoprotein named cytochrome b5 reductase (also known as metHb reductase), and cytochrome b5. The Fe3+ of metHb is reduced back to the Fe2+ state by the action of reduced cytochrome b5:
Hb-Fe3+ + Ñyt b5 red ® Hb-Fe2+ + Ñyt b5 ox
Reduced cytochrome b5 is then regenerated by the action of cytochrome b5 reductase:
Ñyt b5 ox + NADH ® Ñyt b5 red + NAD
Haemoglobin or hemoglobin (frequently abbreviated as Hb or Hgb) is the iron-containing oxygen-transport metalloprotein in the red blood cells of the blood in vertebrates and other animals; in mammals the protein makes up about 97% of the red cell’s drycontent, and around 35% of the total content including water. Hemoglobin transports oxygen from the lungs or gills to the rest of the body, such as to the muscles, where it releases the oxygen load. Hemoglobin also has a variety of other gas-transport and effect-modulation duties, which vary from species to species, and which in invertebrates may be quite diverse.
The name hemoglobin is the concatenation of heme and globin, reflecting the fact that each subunit of hemoglobin is a globular protein with an embedded heme (or haem) group; each heme group contains an iron atom, and this is responsible for the binding of oxygen. The most common type of hemoglobin in mammals contains four such subunits, each with one heme group.
Mutations in the genes for the hemoglobin protein in humans result in a group of hereditary diseases termed thehemoglobinopathies, the most common members of which are sickle-cell disease and thalassemia. Historically in human medicine, thehemoglobinopathy of sickle-cell disease was the first disease to be understood in its mechanism of dysfunction, completely down to the molecular level. However, not all of such mutations produce disease states, and are formally recognized as hemoglobin variants (not diseases).
Hemoglobin (Hb) is synthesized in a complex series of steps. The heme portion is sythesized in both the the mitochondria andcytosol of the immature red blood cell, while the globin protein portions of the molecule are sythesized by ribosomes in the cytosol. Production of Hb continues in the cell throughout its early development from the proerythroblast to the reticulocyte in the bone marrow. At this point, the nucleus is lost in mammals, but not in birds and many other species. Even after the loss of the nucleus in mammals, however, residual ribosomal RNA allows further synthesis of Hb until the reticulocyte loses its RNA soon after entering the vasculature (this hemoglobin-synthetic RNA in fact gives the reticulocyte its reticulated appearance and name).
The empirical chemical formula of the most common human hemoglobin is C2952H4664N812O832S8Fe4, but as noted above,hemoglobins vary widely across species, and even (through common mutations) slightly among subgroups of humans.
In humans, the hemoglobin molecule is an assembly of four globular protein subunits. Each subunit is composed of a protein chain tightly associated with a non-protein heme group. Each protein chain arranges into a set of alpha-helix structural segments connected together in a globin fold arrangement, so called because this arrangement is the same folding motif used in otherheme/globin proteins such as myoglobin. This folding pattern contains a pocket which strongly binds the heme group.
A heme group consists of an iron (Fe) atom held in a heterocyclic ring, known as a porphyrin. The iron atom, which is the site of oxygen binding, bonds with the four nitrogens in the center of the ring, which all lie in one plane. The iron is also bound strongly to the globular protein via the imidazole ring of a histidine residue below the porphyrin ring. A sixth position can reversibly bind oxygen, completing the octahedral group of six ligands. Oxygen binds in an "end-on bent" geometry where one oxygen atom binds Fe and the other protrudes at an angle. When oxygen is not bound, a very weakly bonded water molecule fills the site, forming a distorted octahedron.
The iron atom may either be in the Fe2+ or Fe3+ state, but ferrihemoglobin (methemoglobin) (Fe3+) cannot bind oxygen. In binding, oxygen temporarily oxidizes Fe to (Fe3+), so iron must exist in the +2 oxidation state in order to bind oxygen. The body reactivates hemoglobin found in the inactive (Fe3+) state by reducing the iron center.
In adult humans, the most common hemoglobin type is a tetramer (which contains 4 subunit proteins) called hemoglobin A, consisting of two α and two β subunits non-covalently bound, each made of 141 and 146 amino acid residues, respectively. This is denoted as α2β2. The subunits are structurally similar and about the same size. Each subunit has a molecular weight of about 17,000daltons, for a total molecular weight of the tetramer of about 68,000 daltons. Hemoglobin A is the most intensively studied of the hemoglobin molecules.
The four polypeptide chains are bound to each other by salt bridges, hydrogen bonds, and hydrophobic interactions. There are two kinds of contacts between the α and β chains: α1β1 and α1β2.
Oxyhemoglobin is formed during respiration when oxygen binds to the heme component of the protein hemoglobin in red blood cells. This process occurs in the pulmonary capillaries adjacent to the alveoli of the lungs. The oxygen then travels through the blood stream to be dropped off at cells where it is utilized in aerobic glycolysis and in the production of ATP by the process of oxidativephosphorylation. It doesn't however help to counteract a decrease in blood pH. Ventilation, or breathing, may reverse this condition by removal of carbon dioxide, thus causing a shift up in pH.
Deoxyhemoglobin is the form of hemoglobin without the bound oxygen. The absorption spectra of oxyhemoglobin anddeoxyhemoglobin differ. The oxyhemoglobine has significantly lower absorption of the 660 nm wavelength than deoxyhemoglobin, while at 940 nm its absorption is slightly higher. This difference is used for measurement of the amount of oxygen in patient's blood by an instrument called pulse oximeter.
Iron's oxidation state in oxyhemoglobin.
The oxidation state of iron in hemoglobin is always +2. It does not change when oxygen binds to the deoxy- form.
Assigning oxygenated hemoglobin's oxidation state is difficult because oxyhemoglobin is diamagnetic (no net unpaired electrons), but the low-energy electron configurations in both oxygen and iron are paramagnetic. Triplet oxygen, the lowest energy oxygen species, has two unpaired electrons in antibonding π* molecular orbitals. Iron(II) tends to be in a high-spin configuration where unpaired electrons exist in eg antibonding orbitals. Iron(III) has an odd number of electrons and necessarily has unpaired electrons. All of these molecules are paramagnetic (have unpaired electrons), not diamagnetic, so an unintuitive distribution of electrons must exist to induce diamagnetism.
The three logical possibilities are:
1) Low-spin Fe2+ binds to high-energy singlet oxygen. Both low-spin iron and singlet oxygen are diamagnetic.
2) High-spin Fe3+ binds to .O2- (the superoxide ion) and antiferromagnetism oppositely aligns the two unpaired electrons, giving diamagnetic properties.
3) Low-spin Fe4+ binds to O22-. Both are diamagnetic.
X-ray photoelectron spectroscopy suggests that iron has an oxidation state of approximately 3.2 and infrared stretching frequencies of the O-O bond suggests a bond length fitting with superoxide. The correct oxidation state of iron is thus the +3 state with oxygen in the -1 state. The diamagnetism in this configuration arises from the unpaired electron on superoxide aligningantiferromagnetically in the opposite direction from the unpaired electron on iron. The second choice being correct is not surprising because singlet oxygen and large separations of charge are both unfavorably high-energy states. Iron's shift to a higher oxidation state decreases the atom's size and allows it into the plane of the porphyrin ring, pulling on the coordinated histidine residue and initiating the allosteric changes seen in the globulins. The assignment of oxidation state, however, is only a formalism so all three models may contribute to some small degree.
Early postulates by bioinorganic chemists claimed that possibility (1) (above) was correct and that iron should exist in oxidation state II (indeed iron oxidation state III as methemoglobin, when not accompanied by superoxide .O2- to "hold" the oxidation electron, is incapable of binding O2). The iron chemistry in this model was elegant, but the presence of singlet oxygen was never explained. It was argued that the binding of an oxygen molecule placed high-spin iron(II) in an octahedral field of strong-field ligands; this change in field would increase the crystal field splitting energy, causing iron's electrons to pair into the diamagnetic low-spin configuration.
Binding of ligands
Binding and release of ligands induces a conformational (structural) change in hemoglobin. Here, the binding and release of oxygen illustrates the structural differences between oxy- and deoxyhemoglobin, respectively. Only one of the four heme groups is shown.
As discussed above, when oxygen binds to the iron center it causes contraction of the iron atom, and causes it to move back into the center of the porphyrin ring plane (see moving diagram). At the same time, the porphyrin ring plane itself is pushed away from the oxygen and toward the imidizole side chain of the histidine residue interacting at the other pole of the iron. The interaction here forces the ring plane sideways toward the outside of the tetramer, and also induces a strain on the protein helix containing thehistidine, as it moves nearer the iron. This causes a tug on this peptide strand which tends to open up heme units in the remainder of the molecule, so that there is more room for oxygen to bind at their heme sites.
In the tetrameric form of normal adult hemoglobin, the binding of oxygen is thus a cooperative process. The binding affinity of hemoglobin for oxygen is increased by the oxygen saturation of the molecule, with the first oxygens bound influencing the shape of the binding sites for the next oxygens, in a way favorable for binding. This positive cooperative binding is achieved through stericconformational changes of the hemoglobin protein complex as discussed above, i.e. when one subunit protein in hemoglobin becomes oxygenated, this induces a conformational or structural change in the whole complex, causing the other subunits to gain an increased affinity for oxygen. As a consequence, the oxygen binding curve of hemoglobin is sigmoidal, or S-shaped, as opposed to the normal hyperbolic curve associated with noncooperative binding.
Hemoglobin's oxygen-binding capacity is decreased in the presence of carbon monoxide because both gases compete for the same binding sites on hemoglobin, carbon monoxide binding preferentially in place of oxygen. Carbon dioxide occupies a different binding site on the hemoglobin. Through the enzyme carbonic anhydrase, carbon dioxide reacts with water to give carbonic acid, which decomposes into bicarbonate and protons:
CO2 + H2O → H2CO3 → HCO3- + H+
The sigmoidal shape of hemoglobin's oxygen-dissociation curve results from cooperative binding of oxygen to hemoglobin.
Hence blood with high carbon dioxide levels is also lower in pH (more acidic). Hemoglobin can bind protons and carbon dioxide which causes a conformational change in the protein and facilitates the release of oxygen. Protons bind at various places along the protein, and carbon dioxide binds at the alpha-amino group forming carbamate. Conversely, when the carbon dioxide levels in the blood decrease (i.e., in the lung capillaries), carbon dioxide and protons are released from hemoglobin, increasing the oxygen affinity of the protein. This control of hemoglobin's affinity for oxygen by the binding and release of carbon dioxide and acid, is known as the Bohr effect.
The binding of oxygen is affected by molecules such as carbon monoxide (CO) (for example from tobacco smoking, cars and furnaces). CO competes with oxygen at the heme binding site. Hemoglobin binding affinity for CO is 200 times greater than its affinity for oxygen, meaning that small amounts of CO dramatically reduces hemoglobin's ability to transport oxygen. When hemoglobin combines with CO, it forms a very bright red compound called carboxyhemoglobin. When inspired air contains CO levels as low as 0.02%, headache and nausea occur; if the CO concentration is increased to 0.1%, unconsciousness will follow. In heavy smokers, up to 20% of the oxygen-active sites can be blocked by CO.
In similar fashion, hemoglobin also has competitive binding affinity for cyanide (CN-), sulfur monoxide (SO), nitrogen dioxide (NO2), and sulfide (S2-), including hydrogen sulfide (H2S). All of these bind to iron in heme without changing its oxidation state, but they nevertheless inhibit oxygen-binding, causing grave toxicity.
The iron atom in the heme group must be in the Fe2+ oxidation state to support oxygen and other gases' binding and transport. Oxidation to Fe3+ state converts hemoglobin into hemiglobin or methemoglobin (pronounced "MET-hemoglobin"), which cannot bind oxygen. Hemoglobin in normal red blood cells is protected by a reduction system to keep this from happening. Nitrogen dioxide and nitrous oxide are capable of converting a small fraction of hemoglobin to methemoglobin, however this is not usually of medical importance (nitrogen dioxide is poisonous by other mechanisms, and nitrous oxide is routinely used in surgical anesthesia in most people without undue methemoglobin buildup).
In people acclimated to high altitudes, the concentration of 2,3-bisphosphoglycerate (2,3-BPG) in the blood is increased, which allows these individuals to deliver a larger amount of oxygen to tissues under conditions of lower oxygen tension. This phenomenon, where molecule Y affects the binding of molecule X to a transport molecule Z, is called a heterotropic allosteric effect.
A variant hemoglobin, called fetal hemoglobin (HbF, α2γ2), is found in the developing fetus, and binds oxygen with greater affinity than adult hemoglobin. This means that the oxygen binding curve for fetal hemoglobin is left-shifted (i.e., a higher percentage of hemoglobin has oxygen bound to it at lower oxygen tension), in comparison to that of adult hemoglobin. As a result, fetal blood in the placenta is able to take oxygen from maternal blood.
Hemoglobin also carries nitric oxide in the globin part of the molecule. This improves oxygen delivery in the periphery and contributes to the control of respiration. NO binds reversibly to a specific cystein residue in globin; the binding depends on the state (R or T) of the hemoglobin. The resulting S-nitrosylated hemoglobin influences various NO-related activities such as the control of vascular resistance, blood pressure and respiration. NO is released not in the cytoplasm of erythrocytes but is transported by an anion exchanger called AE1 out of them.
Degradation of hemoglobin in vertebrate animals
When red cells reach the end of their life due to aging or defects, they are broken down, the hemoglobin molecule is broken up and the iron gets recycled. When the porphyrin ring is broken up, the fragments are normally secreted in the bile by the liver. This process also produces one molecule of carbon monoxide for every molecule of heme degraded; this is one of the few natural sources of carbon monoxide production in the human body, and is responsible for the normal blood levels of carbon monoxide even in people breathing pure air. The other major final product of heme degradation is bilirubin. Increased levels of this chemical are detected in the blood if red cells are being destroyed more rapidly than usual. Improperly degraded hemoglobin protein or hemoglobin that has been released from the blood cells too rapidly can clog small blood vessels, especially the delicate blood filtering vessels of the kidneys, causing kidney damage
Role in disease
Decrease of hemoglobin, with or without an absolute decrease of red blood cells, leads to symptoms of anemia. Anemia has many different causes, although iron deficiency and its resultant iron deficiency anemia are the most common causes in the Western world. As absence of iron decreases heme synthesis, red blood cells in iron deficiency anemia are hypochromic (lacking the red hemoglobin pigment) and microcytic (smaller than normal). Other anemias are rarer. In hemolysis (accelerated breakdown of red blood cells), associated jaundice is caused by the hemoglobin metabolite bilirubin, and the circulating hemoglobin can cause renal failure.
Some mutations in the globin chain are associated with the hemoglobinopathies, such as sickle-cell disease and thalassemia. Other mutations, as discussed at the beginning of the article, are benign and are referred to merely as hemoglobin variants.
There is a group of genetic disorders, known as the porphyrias that are characterized by errors in metabolic pathways of hemesynthesis. King George III of the United Kingdom was probably the most famous porphyria sufferer.
To a small extent, hemoglobin A slowly combines with glucose at a certain location in the molecule. The resulting molecule is often referred to as Hb A1c. As the concentration of glucose in the blood increases, the percentage of Hb A that turns into Hb A1cincreases. In diabetics whose glucose usually runs high, the percent Hb A1c also runs high. Because of the slow rate of Hb A combination with glucose, the Hb A1c percentage is representative of glucose level in the blood averaged over a longer time (the half-life of red blood cells, which is typically 50-55 days).
Hemoglobin levels are amongst the most commonly performed blood tests, usually as part of a full blood count or complete blood count. Results are reported in g/L, g/dL or mol/L. For conversion, 1 g/dL is 0,621 mmol/L. If the total hemoglobin concentration in the blood falls below a set point, this is called anemia. Normal values for hemoglobin levels are:
• Women: 12.1 to 15.1 g/dl
• Men: 13.8 to 17.2 g/dl
• Children: 11 to 16 g/dl
• Pregnant women: 11 to 12 g/dl
Anemias are further subclassified by the size of the red blood cells, which are the cells which contain hemoglobin in vertebrates. They can be classified as microcytic (small sized red blood cells), normocytic (normal sized red blood cells), or macrocytic (large sized red blood cells). The hemaglobin is the typical test used for blood donation. A comparison with the hematocrit can be made by multiplying the hemaglobin by three. For example, if the hemaglobin is measured at 17, that compares with a hematocrit of .51.
Glucose levels in blood can vary widely each hour, so one or only a few samples from a patient analyzed for glucose may not be representative of glucose control in the long run. For this reason a blood sample may be analyzed for Hb A1c level, which is more representative of glucose control averaged over a longer time period (determined by the half-life of the individual's red blood cells, which is typically 50-55 days). People whose Hb A1c runs 6.0% or less show good longer-term glucose control. Hb A1c values which are more than 7.0% are elevated. This test is especially useful for diabetics.