haemoglobin Both the red badge of courage and the blue blood of the aristocrat are due to haemoglobin, the pigment that gives blood its colour. Take it away, by removing the blood cells, and the resulting plasma is a very pale yellow. Haemoglobin combines with oxygen, enabling blood to carry 70 times more than if the oxygen were simply dissolved. Animals that are physically active and larger than a pea could scarcely survive without it. ‘But for haemoglobin's existence, man might never have attained any activity which the lobster does not possess, or had he done so, it would have been with a body as minute as the fly's’ ( J. Barcoft).

Haemoglobin, contained in the red cells of the blood and constituting the main site of iron in the body, is present in all vertebrate species. In the human adult it is synthesized in the developing red cells in the bone marrow. Many worms have haemoglobin, but others and also most molluscs have different and more primitive oxygen-carrying pigments, which have not survived into higher forms of evolution.

Haemoglobin not only distributes oxygen as it is required by the tissues but is also an important store of the gas. Healthy humans have about 15 g of haemoglobin per litre of blood, and this can bind with 200 ml of oxygen per litre. With the body at rest the tissues only remove about one-quarter of the available oxygen reaching them in arterial blood, the other three-quarters remaining in the venous blood returning to the lungs. This constitutes an important reserve of oxygen supply which can be called on in conditions of work and exercise. In a typical total blood volume of 5 litres, even though more than half is in the veins, we thus have about 0.75 litre of oxygen combined with haemoglobin in the blood, and we have about the same amount as gas in the lungs. If we stop breathing, for example by holding our breath, these stores will maintain the functions of the brain for at the most a few minutes — but without them brain function would cease almost immediately.

The amount of oxygen free in solution in the blood plays no important role in carriage of oxygen to the tissues. The amount depends on the pressure of the gas in the lungs (see figure). If we breathe pure oxygen the amount in solution rises almost seven-fold and it can become a significant contribution to the body. If we were to breathe pure oxygen in a chamber at a pressure of three atmospheres, all the oxygen we need could be carried in solution and we would not need haemoglobin. This treatment is used for some conditions when haemoglobin is seriously deficient, but there are significant hazards of breathing high-pressure oxygen.

Each haemoglobin molecule consists of four iron-containing parts (haems) and four protein chains (globins). The fact that blood contained iron was discovered in 1747 by Menghini, who showed that if blood was burnt to an ash, iron-like particles could be extracted by a magnet. Chemical analysis of haemoglobin began in the mid nineteenth century and culminated in one of the great early triumphs of molecular biology, when in the 1960s the full chemical structure of haemoglobin was worked out.

Each haemoglobin molecule can combine with four oxygen molecules, but with no more. The complete combination is called oxygen saturation. The degree of combination depends on the pressure of the gas; in healthy humans the pressure in the alveoli of the lungs is above that needed for saturation. If the alveolar oxygen pressure is increased, for example by breathing more deeply or by inhaling pure oxygen, the haemoglobin in the blood will not take up any additional oxygen (see figure). However in patients with arterial blood not saturated with oxygen, for example with lung or heart disease, stimulation of breathing or administration of oxygen should increase the oxygen carriage in the blood and be beneficial or life-saving.

The combination of oxygen with haemoglobin is not related linearly to the oxygen pressure, and this is crucially important in its function. As oxygen pressure reduces below that required for full saturation, haemoglobin is relatively little desaturated until and unless the oxygen pressure reaches about the level which blood normally encounters in the oxygen-using tissues: it then parts with it readily. Thus in breath-holding, or in disease, or at altitude, alveolar oxygen pressure can approach half its normal value before haemoglobin saturation declines steeply in the blood leaving the lungs; and saturation is not itself halved until the oxygen pressure is reduced by almost two-thirds. Thus the properties of haemoglobin defend the oxygen supply against interruptions of breathing or shortage of oxygen in the atmosphere, whilst promoting its off-loading around the body.

The combination of haemoglobin and oxygen is weak, and oxygen can be pulled from the blood if the surrounding pressure of oxygen is low; indeed a vacuum will extract all the oxygen from a sample of blood. When blood flows through the capillaries of tissues which are using oxygen for metabolism, the low oxygen pressure in the tissue cells draws oxygen from its combination with haemoglobin and the gas flows into the cells. The resulting venous blood contains less than its full oxygen saturation, and the haemoglobin is partly ‘deoxygenated’. Such haemoglobin does not have the bright red colour of saturated haemoglobin, but is more blue. Thus conventionally arterial blood is red and venous blood is blue. In cyanosis tissues are bluish because their blood is deficient in oxygen.

Haemoglobin can also combine with carbon dioxide to form carbaminohaemoglobin, and this is one way in which this gas is carried round the body. The two gases have a complex chemical interaction with haemoglobin. When in metabolizing tissues carbon dioxide enters the blood, its combination with haemoglobin results in a weaker affinity for oxygen, which is split off and enters the cells. The reverse happens in the lungs. Temperature has a similar effect: if local temperature rises oxyhaemoglobin splits more easily. Both mechanisms help to match gas exchange to changing activity.

Red cells also contain 2,3-diphosphoglycerate (DPG), a substance that increases the readiness with which haemoglobin gives up its oxygen. The DPG is increased in exercise and at high altitude, which facilitates the supply of oxygen to the tissues. Unfortunately this process takes several hours. Stored blood loses its DPG and is therefore less effective on transfusion than fresh blood, although there are ways to treat it that restore the DPG.

Although the haem is the essential part of the haemoglobin molecule to enable it to combine with oxygen, it is the four globin molecules which determine the amount of binding or affinity for haemoglobin and oxygen. The globins are identified by Greek letters, and a very large number have been discovered, many of them related to diseases of the blood. Healthy human adults have two a-globins and two b-globins. Fetuses have two a-and two g-globins. As a result fetal haemoglobin has a stronger affinity for oxygen than does the adult form. When maternal blood flows through the placental circulation, oxygen diffuses across the placental barrier into the fetus and, because of the difference between the two haemoglobins, the fetus extracts a proportionally higher amount of oxygen. This success in parasitism is clearly to the advantage of the fetus. After birth the fetal haemoglobin is slowly replaced by the adult version.

In healthy humans, haemoglobin is only found in erythrocytes, the blood red cells. The advantage in confining the haemoglobin in cells is threefold. First, if the haemoglobin were free in solution it would give the blood a treacle-like consistency, and the heart would be unable to force it fast enough through the capillaries. Second, the chemical environment in the red cell, including for example the presence of DPG, allows the haemoglobin to take up and release oxygen with greatest efficiency. And third, if the haemoglobin were free in solution it would be excreted and lost in the kidneys. Patients with red cell breakdown, for example in malaria, pass haemoglobin into the urine, where it is broken down to the brown pigment methaemoglobin; hence one form of malaria is called ‘black-water fever’.

A few animals — some of the worms mentioned earlier — do have free oxygen-carrying pigments in the blood, but their molecular sizes are 40 times that of haemoglobin, so they are not excreted. One species of antarctic fish is said to lack both red cells and haemoglobin, but it lives in a cold environment and its metabolism and oxygen requirement must be very low.

Human red cells live on average about 120 days in the bloodstream, and then they become fragile and are broken up, especially by scavenger cells in the spleen and liver. The haemoglobin is not released into the blood, but is immediately broken down into haem and globins. The haem is in turn split into iron, which forms chemical compounds as part of the blood iron pool available for future haemoglobin synthesis, and an amber pigment, bilirubin, which contributes to the pale colour of plasma. Bilirubin combines with albumin in the blood, and the large size of this combined molecule prevents it from being excreted in the kidneys. Instead it passes to the liver, where it is excreted in the bile, contributing to its colour. When it reaches the intestines it is acted on by bacterial flora, and forms the brown pigment stercobilinogen. Most of the stercobilinogen appears in the faeces, giving it its characteristic colour (but not its odour), and the rest is reabsorbed into the bloodstream. Here a proportion recirculates in the bile but most, now called urobilinogen, is excreted in the urine. Thus not only does haemoglobin provide the colour of the blood, but its breakdown products are largely responsible for the colours of plasma, bile, faeces, and urine. Jaundice is due to an excess of bilirubin in the blood and tissues.

There are many diseases caused by abnormal haemoglobins. In all of them it is the globin part of the molecule which is abnormal. Not only may haemoglobin be unable to combine normally with oxygen, but since the haemoglobin is an integral part of the structure of the red cell, these cells may be deformed. An example is sickle cell disease, where the red cells become rigid and deformed and break down more readily, leading to anaemia. Another common disease is thalassaemia, where there is a defect in the synthesis of the b-globin chains. Less common conditions are the persistence of fetal haemoglobin long after birth, and abnormalities in the enzymes associated with haemoglobin (e.g. DPG) that affect its affinity for oxygen.

John Widdicombe

Hemoglobin

Hemoglobin is a protein formed of two subunits (alpha and beta) that are found in red blood cells. The protein functions to pick up oxygen and distribute it throughout the body.

Both the alpha and beta subunits need to be present for the acquisition of oxygen, as does an iron molecule. Indeed, it is the presence of the iron that gives red blood cells the distinctive color that inspired their name.

The presence of iron enables hemoglobin to alternatively bind oxygen and carbon dioxide. As blood is pumped through the myriad of tiny channels that permeate the lung, oxygen can diffuse across the membrane of the channel to the red blood cell-containing fluid within the channels. There, the binding of oxygen to the iron-containing hemoglobin occurs. The oxygenated red blood cells pass out of the lungs and circulate throughout the body, transporting the oxygen along with them to cells.

Once oxygen has been released from hemoglobin, the vacated binding site is able to bind carbon dioxide and other waste products of cellular metabolism. This process is vital to maintain a body in a proper equilibrium. If otherwise allowed to accumulate, these products would reach toxic concentrations. The hemoglobin bound carbon dioxide is transported back to the lungs, where it is released from the red blood cells and expired. Completing the cycle, the once-aging vacant iron site can bind another molecule of oxygen.

The alpha and beta subunits of hemoglobin are encoded by separate genes. Normally, an individual has four alpha-encoding genes and two beta-encoding genes. Despite the different number of genes, protein production is coordinated so that precisely equal amounts of the subunits are made during red blood cell manufacture. The subunits are incorporated into the developing blood cells, where they remain throughout the days-to-weeks lifespan of the cells.

In the majority of people, both hemoglobin itself and the genes encoding the subunits are invariant. This aspect would seemingly rule out the routine use of hemoglobin as a tool to identify someone in a forensic investigation. However, in people with sickle cell disease (in which the abnormally-shaped red blood cell cannot easily pass through all blood vessels, producing an oxygen shortage) and thalassemia (a group of related maladies, in which hemoglobin production is low) the mutated hemoglobin gene that is the root of the malady can be detected in now-routine molecular biological test procedures such as gene sequencing (where the order of the bases that make up a gene is determined).

As a component of blood, hemoglobin can be an important facet of a forensic investigation, especially to help detect the illegal practice known as “blood doping” in sports, and in helping to identify if a blood sample was from someone with a blood abnormality such as sickle cell disease.

In addition to the well-known hemoglobin disorders that underlie sickle cell anemia and thalassemia, there are several hundred other forms of abnormal hemoglobin. These forms, which usually do not cause harm to a person, can be detected using specialized molecular examination techniques, and so can be useful forensically.

In the case of a blood stain at a crime or accident scene, determination of the amount of hemoglobin can be useful in indicating the approximate age of a person as well as their sex. Hemoglobin content can be determined in a less sophisticated fashion than hemoglobin disorders. Blood cells are broken apart in automated blood analyzers to free the hemoglobin. Upon exposure to a cyanide-containing compound, free hemoglobin binds the cyanide. The resulting compound (cyanmethemoglobin) specifically absorbs light at a wavelength of 540 nanometers, permitting the amount of hemoglobin to be determined. Normal ranges for hemoglobin (expressed as grams per deciliter; a deciliter being 100 milliliters) are 17–22 for newborns, 11–13 for children, 14–18 for adult males and 12–16 for adult females, as a few examples. While not by itself definitive, hemoglobin content determinations of a blood sample can be another useful piece of forensic evidence.

When hemoglobin levels in blood or the red blood cells are low, as in the aforementioned cases of sickle cell anemia and thalassemia, an individual is described as being anemic. Anemia can also arise from loss of blood in a traumatic injury or internal blood loss, a nutritional deficiency, or compromised bone marrow. Thus, hemoglobin analysis can provide clues concerning the health of a victim or suspect.

Higher than normal levels of hemoglobin can be encountered in people who routinely live or work at high altitude, due to the increased production of red blood cells to maximize the blood’s oxygen carrying capacity. Athletes who have artificially increased this capacity through blood doping by infusing their own previously collected red blood cells, or injecting the drug erythropoetin, which triggers the body to increase its red blood cell supply, can be found out in this way.

See also Blood; Hemophilia.

Brian Hoyle

Hemoglobin

As a component of blood , hemoglobin can be an important facet of a forensic investigation, especially to help detect the illegal practice known as "blood doping" in sports, and in helping to identify if a blood sample was from someone with a blood abnormality such as sickle cell disease.

Hemoglobin is a protein formed of two subunits (alpha and beta) that is found in red blood cells. The protein functions to pick up oxygen and distribute it throughout the body.

Both the alpha and beta subunits need to be present for the acquisition of oxygen, as does an iron molecule. Indeed, it is the presence of the iron that gives red blood cells the distinctive color that inspired their name.

The presence of iron enables hemoglobin to alternatively bind oxygen and carbon dioxide. As blood is pumped through the myriad of tiny channels that permeate the lung, oxygen can diffuse across the membrane of the channel to the red blood cell-containing fluid within the channels. There, the binding of oxygen to the iron-containing hemoglobin occurs. The oxygenated red blood cells pass out of the lungs and circulate throughout the body, transporting the oxygen along with them to cells.

Once oxygen has been released from hemoglobin, the vacated binding site is able to bind carbon dioxide and other waste products of cellular metabolism. This process is vital to maintain a body in a proper equilibrium. If otherwise allowed to accumulate, these products would reach toxic concentrations. The hemoglobin bound carbon dioxide is transported back to the lungs, where it is released from the red blood cells and expired. Completing the cycle, the once-aging vacant iron site can bind another molecule of oxygen.

The alpha and beta subunits of hemoglobin are encoded by separate genes. Normally, an individual has four alpha-encoding genes and two beta-encoding genes. Despite the different number of genes, protein production is coordinated so that precisely equal amounts of the subunits are made during red blood cell manufacture. The subunits are incorporated into the developing blood cells, where they remain throughout the days-to-weeks lifespan of the cells.

In the majority of people, both hemoglobin itself and the genes encoding the subunits are invariant. This aspect would seemingly rule out the routine use of hemoglobin as a tool to identify someone in a forensic investigation. However, in people with sickle cell disease (in which the abnormally-shaped red blood cell cannot easily pass through all blood vessels, producing an oxygen shortage) and thalassemia (a group of related maladies, in which hemoglobin production is low) the mutated hemoglobin gene that is the root of the malady can be detected in now-routine molecular biological test procedures such as gene sequencing (where the order of the bases that make up a gene is determined).

If a blood sample recovered from the investigation scene contains a mutated hemoglobin gene, the discovery of the same mutation in a blood sample of a suspect, for example, can be powerful, although not unequivocal, evidence tying the suspect to the crime scene.

In addition to the well-known hemoglobin disorders that underlie sickle cell anemia and thalassemia, there are several hundred other forms of abnormal hemoglobin. These forms, which usually do not cause harm to a person, can be detected using specialized molecular examination techniques, and so can be useful forensically.

In the case of a bloodstain at a crime or accident scene, determination of the amount of hemoglobin can be useful in indicating the approximate age of a person as well as their sex. Hemoglobin content can be determined in a less sophisticated fashion than hemoglobin disorders. Blood cells are broken apart in automated blood analyzers to free the hemoglobin. Upon exposure to a cyanide-containing compound, free hemoglobin binds the cyanide. The resulting compound (cyanmethemoglobin) specifically absorbs light at a wavelength of 540 nanometers, permitting the amount of hemoglobin to be determined. Normal ranges for hemoglobin (expressed as grams per deciliter; a deciliter being 100 milliliters) are 17–22 for newborns, 11–13 for children, 14–18 for adult males and 12–16 for adult females, as a few examples. While not by itself definitive, hemoglobin content determinations of a blood sample can be another useful piece of forensic evidence.

When hemoglobin levels in blood or the red blood cells are low, as in the aforementioned cases of sickle cell anemia and thalassemia, an individual is described as being anemic. Anemia can also arise from loss of blood in a traumatic injury or internal blood loss, a nutritional deficiency, or compromised bone marrow. Thus, hemoglobin analysis can provide clues concerning the health of a victim or suspect.

Higher than normal levels of hemoglobin can be encountered in people who routinely live or work at high altitude, due to the increased production of red blood cells to maximize the blood's oxygen carrying capacity. Athletes who have artificially increased this capacity through blood doping by infusing their own previously collected red blood cells, or injecting the drug erythropoetin, which triggers the body to increase its red blood cell supply, can be found out in this way.

see also Blood.

Hemoglobin

Hemoglobin is an iron-containing protein found in the blood of nearly all vertebrates and many invertebrates . It transports oxygen from the lungs or gills of an animal to the tissues. Four polypeptide chains, each wrapped in a specific way around its own heme group , make up the hemoglobin molecule. An iron atom in the ferrous state (Fe²⁺) sits in the middle of the heme. Ferric hemoglobin (Fe³⁺) cannot bind oxygen.

The polypeptide chains of hemoglobin are of two kinds; in the hemoglobin of an adult human, HbA, they are designated as α and β, and the subunit structure of HbA is written α ₂β ₂. The α chain contains 141 amino acid residues, the β chain 146. Throughout most of the animal kingdom the subunit structure of hemoglobin remains the same: two polypeptides of one kind with 141 amino acid residues and two of another with 146 residues. The amino acid sequences (the primary structure) of the polypeptides, however, are species dependent. Closely related animals display fewer differences in these amino acid sequences. The α and β chains of human hemoglobin, for example, differ from chicken hemoglobin in 35 and 45 places, respectively. Between human and horse α and β chains these differences drop to 18 and 25. As for chimpanzee and human, their hemoglobins are identical.

Often several kinds of hemoglobin exist in a given animal. In the adult human, for example, HbA (α ₂δ ₂) makes up 98 percent of the total hemoglobin, and HbA₂ (α ₂δ ₂), the remaining 2 percent. The polypeptide chain δ has the same number of amino acids as the β chain, but the sequences differ in 10 places. A third kind of human hemoglobin, fetal hemoglobin or HbF (α ₂γ ₂), constitutes over 80 percent of the total hemoglobin of a newborn, but vanishes rapidly during the first year of life. The amino acid sequences of the γ and β chains differ in 39 of the 146 residues. Additional types of human hemoglobins exist at embryonic stages of life.

Each polypeptide chain of a hemoglobin molecule coils into several helical segments (the secondary structure of the polypeptide chain) that are linked with nonhelical segments. Intertwining sets of helices wrap tightly around the heme group and produce a compact subunit (the tertiary structure). The four subunits are packed into a nearly spherical package (the quaternary structure) of 55-angstrom (2.17 ×10⁻⁷-inch) diameter.

Each ferrous iron within hemoglobin provides one binding site for O₂. Thus a single hemoglobin molecule has the capacity to combine with four molecules of oxygen. Hemoglobin binds oxygen in a cooperative fashion; occupation of one binding site enhances the affinity of another binding site for oxygen in the molecule. Consequently, the oxygenation curve of hemoglobin (see Figure 1), in which the fractional saturation of hemoglobin with oxygen is displayed as a function of the oxygen pressure in the alveoli of the lungs, rises slowly at first, then more steeply, until it levels off and approaches unity (100% saturation). The steep rise in the oxygenation curve over a relatively small interval of oxygen pressure allows hemoglobin to serve as an efficient transporter of oxygen.

see also Primary Structure; Quaternary Structure; Secondary Structure; Tertiary Structure; Transport Protein.

N. M. Senozan

Bibliography

Dickerson, R. E., and Geis, I. (1983). Hemoglobin: Structure, Function and Evolution. Menlo Park, CA: Benjamin/Cummings.

Horton, H. R.; Moran, L. A.; Ochs, R. S.; Rawn, J. D.; and Scrimgeour, K. G. (2002). Principles of Biochemistry, 3rd edition. Upper Saddle River, NJ: Prentice Hall.

haemoglobin One of a group of globular proteins occurring widely in animals as oxygen carriers in blood. Vertebrate haemoglobin comprises two pairs of polypeptide chains, known as α-chains and β-chains (forming the globin protein), with each chain folded to provide a binding site for a haem group. Each of the four haem groups binds one oxygen molecule to form oxyhaemoglobin. Dissociation occurs in oxygen-depleted tissues: oxygen is released and haemoglobin is reformed (see Bohr effect; haemoglobinic acid; oxygen dissociation curve). The haem groups also bind other inorganic molecules, including carbon monoxide (to form carboxyhaemoglobin). In vertebrates, haemoglobin is contained in the red blood cells (erythrocytes).