Hemoglobin

Hemoglobin or haemoglobin (frequently abbreviated as Hb) is the iron-containing oxygen-transport metalloprotein in the red cells of the blood in mammals and other animals. Hemoglobin transports oxygen from the lungs to the rest of the body, such as to the muscles, where it releases the oxygen load.

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 types of hemoglobin contains four such subunits, each with one heme group.

Mutations in the gene for the hemoglobin protein result in a group of hereditary diseases termed the hemoglobinopathies, the most common members of which are sickle-cell disease and thalassemia.

Structure


Heme group

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 individual protein chain arranges in a set of alpha-helix structural segments connected together in a "myoglobin fold" arrangement, so called because this arrangement is the same folding motif used in the heme/globin proteins. This folding pattern contains a pocket which is suitable to strongly bind the heme group.

A heme group consists of an iron atom held in a heterocyclic ring, known as a porphyrin. This iron atom is the site of oxygen binding. The iron atom is bonded equally to all four nitrogens in the center of the ring, which lie in one plane. Two additional bonds perpendicular to the plane on each side can be formed with the iron to form the fifth and sixth positions, one connected strongly to the protein, the other available for binding of oxygen. The iron atom can either be in the Fe2+ or Fe3+ state, but ferrihaemoglobin (Methaemoglobin) (Fe3+) cannot bind oxygen.

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 16,000 daltons, for a total molecular weight of the tetramer of about 64,000 daltons. Haemoglobin A is the most intensively studied of the haemoglobin molecules.

The four polypeptide chains are bound to each other by salt bridges, hydrogen bonds and hydrophobic interaction. There are two kinds of contacts between the α and β chains: α1β1 and α1β2.

Binding of ligands

In the tetrameric form of normal adult hemoglobin, the binding of oxygen is a cooperative process. The binding affinity of hemoglobin for oxygen is increased by the oxygen saturation of the molecule. 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. This positive cooperative binding is achieved through steric conformational changes of the hemoglobin protein complex: When one subunit protein in hemoglobin becomes oxygenated, it induces a conformational or structural change in the whole complex causing the other subunits to gain an increased affinity for oxygen.

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 α-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, α2β2), 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.

Degradation of hemoglobin

When red cells reach the end of their life due to aging or defects, they are broken down, and the hemoglobin molecule broken up and the iron recycled. When the porphyrin ring is broken up, the fragments are normally secreted in the bile by the liver. The 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 can clog small blood vessels, especially the delicate blood filtering vessels of the kidneys, causing kidney damage.

Role in disease

Decreased levels 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.

Mutations in the globin chain are associated with the hemoglobinopathies, such as sickle-cell disease and thalassemia.

There is a group of genetic disorders, known as the porphyrias that are characterized by errors in metabolic pathways of heme synthesis. 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 A1c increases. 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).

Diagnostic use

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 hemoglobin level falls below a set point this is called anemia. Anemias are classified by the size of the red blood cells, which are the cells which contain hemoglobin. They can be classified as microcytic (small sized red blood cells), normocytic (normal sized red blood cells) and macrocytic (large sized red blood cells).

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.

This Hb A1c level is only useful in individuals who have red blood cells (RBCs) with normal survivals (i.e., normal half-life). In individuals with abnormal RBCs, whether due to abnormal hemoglobin molecules (such as Hemoglobin S in Sickle Cell Anemia) or RBC membrane defects - or other problems, the RBC half-life is frequently shortened. In these individuals an alternative test called "fructosamine level" can be used. It measures the degree of glycation (glucose binding) to albumin, the most common blood protein, and reflects average blood glucose levels over the previous 18-21 days, which is the half-life of albumin molecules in the circulation.

Other biological oxygen-binding proteins

Hemoglobin is by no means unique; there are a variety of oxygen transport and binding proteins throughout the animal (and plant) kingdom. Other organisms including bacteria, protozoans and fungi all have hemoglobin-like proteins whose known and predicted roles include the reversible binding of gaseous ligands.

Myoglobin: Found in the muscle tissue of many vertebrates including humans (gives muscle tissue a distinct red or dark gray color). Is very similar to hemoglobin in structure and sequence, but is not arranged in tetramers, it is a monomer and lacks cooperative binding and is used to store oxygen rather than transport it.

Hemocyanin: Second most common oxygen transporting protein found in nature. Found in the blood of many arthropods and molluscs. Uses copper prosthetic group instead of iron heme groups and is blue in color when oxygenated.

Hemerythrin: Some marine invertebrates and a few species of annelid use this iron containing non-heme protein to carry oxygen in their blood. Appears pink/violet when oxygenated, clear when not.

Chlorocruorin: Found in many annelids, and is very similar to Erythrocruorin, but the heme group is significantly different in structure. Appears green when deoxygenated and red when oxygenated.

Vanabins: Also known as Vanadium Chromagen are found in the blood of Sea squirt and are hypothesised to use the rare metal Vanadium as its oxygen binding prosthetic group, but this hypothesis is unconfirmed.

Erythrocruorin: Found in many annelids, including earthworms. Giant free-floating blood protein, contains many dozens even hundreds of Iron heme containing protein subunits bound together into a single protein complex with a molecular masses greater than 3.5 million daltons.

Pinnaglobin: Only seen in the mollusk Pinna squamosa. Brown manganese-based porphyrin protein.

Leghemoglobin: In leguminous plants, such as alfalfa or soybeans, the nitrogen fixing bacteria in the roots are protected from oxygen by this iron heme containing, oxygen binding protein.

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