Hemoglobin

• 1. Introduction

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  Hemoglobin is the iron-bearing protein that fills red blood cells, and nearly every vertebrate depends on it. The lone exception is a single family of fish, the Channichthyidae, which live without it. Chemists also write the name as haemoglobin, or shorten it to Hb or Hgb. In a healthy person, every 100 milliliters of blood holds between 12 and 20 grams of this one protein. That is a remarkable load for a fluid to carry. It raises a string of questions. Why did scientists once laugh at the first estimate of the molecule's size? How does one protein manage to grab oxygen in the lungs and let it go in the tissues? And what happens when another gas takes its place on the iron?

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• 2. A Ridiculed Number

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  In 1825, Johann Friedrich Engelhart noticed that the hemoglobins of several species held iron and protein in the same ratio. From the known atomic mass of iron, he calculated a molecular mass of n times 16,000, where n is the number of iron atoms in each molecule. It was the first time anyone had weighed a protein. Colleagues dismissed it as a "hasty conclusion" and ridiculed it, because they could not believe any molecule could be so large. Exactly a century later, in 1925, Gilbert Smithson Adair measured the osmotic pressure of hemoglobin solutions and confirmed Engelhart's result. Today n is known to be 4.

  Blood had been known to carry oxygen since at least 1794. In 1840, Hünefeld described the oxygen-carrying property of hemoglobin itself. In 1851, the German physiologist Otto Funke published a series of articles about growing hemoglobin crystals. He diluted red blood cells step by step with a solvent such as pure water, alcohol or ether, then let the solvent evaporate slowly from the protein solution. A few years later, Felix Hoppe-Seyler described how the protein takes up oxygen reversibly. The French physiologist Claude Bernard worked out the role the molecule plays in the blood.

  Crystals mattered again once X-ray crystallography made it possible to solve protein structures. In 1959, Max Perutz determined the molecular structure of hemoglobin. He shared the 1962 Nobel Prize in Chemistry with John Kendrew, who had sequenced the globular protein myoglobin. That muscle protein, a close relative of hemoglobin with a very different job, comes back later in this story.

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• 3. Iron and the Switch

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  Hemoglobin A, the main adult form in humans, is assembled from four protein chains: two alpha and two beta. The alpha chains have 141 amino acid residues and the beta chains have 146. Each chain weighs about 16,000 daltons, which puts the whole assembly near 64,000, or 64,458 grams per mole. The name records this design. It joins heme and globin, because every subunit is a globular protein with a heme group embedded in it.

  Most of the amino acids in each chain form alpha helices, joined by short straight stretches. Hydrogen bonds hold the helical sections steady. This arrangement is called the globin fold, and the same pattern appears in other heme proteins such as myoglobin. Its pocket grips the heme tightly. Salt bridges, hydrogen bonds and the hydrophobic effect then bind the four chains to one another.

  At the center of each heme sits one iron ion, held in a ring called a porphyrin. The ring is made of four pyrrole molecules linked in a circle by methine bridges. The iron coordinates with four nitrogen atoms in the ring, all lying in one plane. Below the ring, a histidine labeled F8, known as the proximal histidine, anchors the heme to the protein. The sixth position on the iron binds oxygen reversibly, and when it is empty a very weakly bonded water molecule fills it. Oxygen attaches in an end-on bent geometry, with one atom bound to the iron and the other sticking out at an angle.

  The iron must be in the ferrous, Fe2+, state to bind oxygen. Ferric hemoglobin, called methemoglobin, cannot do it. During binding, oxygen briefly oxidizes the iron while turning into a superoxide ion. Red blood cells carry a reduction system that guards against methemoglobin, and the enzyme methemoglobin reductase can eventually reactivate it. Experimental evidence even suggests the iron behaves as iron(III) in oxyhemoglobin, with the oxygen present as a superoxide anion.

  Each gram of hemoglobin binds 1.34 milliliters of oxygen. That raises the blood's total oxygen capacity seventy-fold compared with oxygen dissolved in plasma alone. In mammals, the protein makes up about 96% of a red blood cell's dry weight and around 35% of its total weight. One molecule can carry up to four oxygen molecules.

  The four binding sites do not act alone. When oxygen lands on one iron, the atom moves into the center of the porphyrin plane. The histidine on the opposite side is dragged toward the ring, which strains the helix that holds it. That strain travels to the other three subunits and makes them easier to load. The molecule shifts from a tense state, called T, to a relaxed state, called R. Because each oxygen helps the next one bind, the binding curve is S-shaped rather than the hyperbolic curve of noncooperative binding.

  Surroundings decide which state wins. In respiring tissues, low pH, high carbon dioxide and high 2,3-BPG favor the tense state, which releases oxygen. In the lungs, high oxygen pressure, high pH and low carbon dioxide favor the relaxed state, which holds oxygen tightly. The S-shaped curve makes the protein efficient at loading and at unloading. Unloading matters just as much, since oxygen that is never released is useless.

  Carbon dioxide reacts with water through the enzyme carbonic anhydrase to give carbonic acid, which splits into bicarbonate and protons. Protons and carbon dioxide bound to hemoglobin lower its appetite for oxygen, a drop known as the Bohr effect. In bony fish, acidity can even cut the total binding capacity, which is called the root effect. Fish also use ATP and GTP, which settle into a phosphate pocket and stabilize the tense state. GTP lowers oxygen affinity far more than ATP, probably because of an extra hydrogen bond.

  Fetal hemoglobin, built from two alpha and two gamma chains, grips oxygen harder than the adult form. Its binding curve sits shifted to the left, so in the placenta the fetal blood can pull oxygen from the mother's. After birth, the gamma chains are gradually replaced by beta chains as the infant grows.

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• 4. Rivals and Recycling

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  Carbon monoxide binds to hemoglobin's heme site with 250 times the affinity of oxygen. Tobacco smoke, exhaust gas and incomplete combustion in furnaces all supply it. The gas is colorless, odorless and tasteless, which is why carbon monoxide detectors became commercially available for homes. The compound it forms with hemoglobin, carboxyhemoglobin, is a very bright red. That color can leave the skin of a poisoning victim looking pink in death rather than white or blue. Air containing just 0.02% carbon monoxide brings headache and nausea, and 0.1% brings unconsciousness. In heavy smokers, up to 20% of the oxygen-active sites can be blocked.

  Cyanide, sulfur monoxide and sulfide, including hydrogen sulfide, also compete for the heme. All of them bind to the iron without changing its oxidation state, yet they block oxygen and cause grave toxicity. Nitric oxide plays a gentler role. It rides on the globin part of the molecule, bound to a thiol group, and is released as oxygen is released. That nitric oxide is thought to widen blood vessels in tissues where oxygen runs low. A transporter in the red cell membrane called AE1 carries it out of the cell. A small fraction of hemoglobin is converted to methemoglobin by nitric oxide, a leftover of a more ancient function of globins.

  The body also makes carbon monoxide itself. When a red blood cell ages or is defective, macrophages in the spleen or liver remove it. Free hemoglobin is cleared from the circulation through a transporter called CD163, found only on monocytes and macrophages. Inside those cells, the molecule is broken up and its iron recycled. Each heme that is degraded yields one molecule of carbon monoxide. This is the only natural source of the gas in the human body, and it accounts for the normal low level in the blood of people breathing clean air.

  The salvaged iron is stored in tissues as hemosiderin or ferritin and travels in plasma on transferrins. The broken porphyrin ring becomes bilirubin, a yellow pigment secreted into the intestines as bile. Intestinal microbes turn it into urobilinogen, and the body finally excretes it in feces as a pigment called stercobilin. Hemoglobin that escapes its cells too fast can clog the delicate filtering vessels of the kidneys. The globin protein itself is broken down into amino acids, which return to the circulation.

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• 5. Genes and Variants

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  On chromosome 16, two neighboring genes called HBA1 and HBA2 code for the alpha subunits of human hemoglobin. The beta subunit comes from a gene called HBB on chromosome 11. Sequences drift apart as species diverge. The most common hemoglobin sequences in humans, bonobos and chimpanzees are completely identical. Gorilla hemoglobin differs from the human version by one amino acid in each of the alpha and beta chains, and the gaps widen between more distant species.

  Mutations in these genes produce variants within a single species. Many are harmless, but some cause hereditary diseases called hemoglobinopathies. The best known is sickle-cell disease, the first human disease whose mechanism was understood at the molecular level. A separate group, the thalassemias, involves underproduction of normal and sometimes abnormal hemoglobins because of problems in how globin genes are regulated. All of these conditions cause anemia.

  The cast of human variants is long. Hemoglobin S, the sickle form, carries a change in the beta chain gene that makes red blood cells sickle. Hemoglobins C and E also change the beta chain and cause a mild chronic hemolytic anemia. Hemoglobin AS, with one normal gene and one sickle gene, gives sickle cell trait. Hemoglobin H, a tetramer of four beta chains, and Hemoglobin Barts, a tetramer of four gamma chains, can appear in variants of alpha thalassemia.

  Other variants are part of normal development. Embryos make forms named Gower 1, Gower 2, Portland I and Portland II. At birth, hemoglobin A makes up over 95% of the total, and hemoglobin A2, with delta chains that begin to form late in the third trimester, has a normal adult range of 1.5-3.5%. In adults, fetal hemoglobin is restricted to a small population of red cells called F-cells, though its level can rise in people with sickle-cell disease or beta-thalassemia.

  Heme and globin are built separately. The heme part is made in the mitochondria and cytosol of immature red blood cells, while ribosomes in the cytosol assemble the globin. Production continues from the proerythroblast stage through the reticulocyte in the bone marrow. Mammalian red cells then lose their nucleus, though birds and many other species keep theirs. Leftover ribosomal RNA keeps the reticulocyte producing hemoglobin for a short time, and that RNA gives the cell its reticulated look and its name.

  A group of genetic disorders called the porphyrias arises from errors in the pathway that builds heme. King George III of the United Kingdom was probably the most famous sufferer.

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• 6. Evolution and Altitude

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  Roughly 450 to 500 million years ago, after jawless fish split from the ancestors of jawed vertebrates, the genes for the alpha and beta subunits arose through a duplication event. Reconstructions of ancestral proteins suggest that the ancestor before the split was a dimer of identical globin subunits. It later evolved into the four-part assembly. Having distinct subunits allowed cooperative oxygen binding and regulation from other sites on the protein. An earlier split, after lampreys diverged from jawed vertebrates, separated myoglobin from hemoglobin. Myoglobin took on oxygen storage while hemoglobin took on transport. The alpha gene later duplicated again to give HBA1 and HBA2, and further duplications produced forms active at different stages of development.

  The lineage reaches even into plants. Leghemoglobin shares only about 15% of its sequence with hemoglobin, which could be chance. But the three-dimensional structures are so alike, and the positions of introns in the two genes so well conserved, that a common origin is the accepted explanation.

  Some of the clearest evolution happens at altitude, where thin air lowers the pressure that pushes oxygen onto hemoglobin. Deer mice from high and low elevations proved nearly identical genetically, except in genes governing the oxygen-carrying capacity of their hemoglobin. Mutations in four genes can account for the difference, and highland mice use oxygen more efficiently. People acclimated to altitude raise their blood levels of 2,3-BPG, which lets them deliver more oxygen under low oxygen tension. Hemoglobin in high-altitude organisms also binds 2,3-BPG less strongly, which tips it toward the relaxed state.

  Andean hummingbirds make an especially striking case. These birds burn energy fast and need a great deal of oxygen, yet they thrive high in the mountains. Mutations in the hemoglobin genes of several high-elevation species, including members of the genus Oreotrochilus, weaken the protein's affinity for inositol hexaphosphate. That molecule plays a role in birds similar to 2,3-BPG in humans, and the change lets the protein bind oxygen at lower pressures. Birds' circulatory lungs reinforce the effect.

  In Tibet, women living at 4,000 meters who carry genotypes with high oxygen saturation have a higher rate of offspring survival. Natural selection seems to be the main force. The exact genotype and mechanism remain unclear. Cold selected too: mammoth hemoglobin carried mutations that delivered oxygen at lower temperatures, which let mammoths move into higher latitudes during the Pleistocene. Most ice fish of the family Channichthyidae went further and lost their hemoglobin genes entirely, as an adaptation to cold water.

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• 7. Cousins and Oddities

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  The giant tube worm, Riftia pachyptila, can reach 2.4 meters in length and populates ocean volcanic vents. It has no digestive tract. Instead, bacteria make up half its weight, oxidizing hydrogen sulfide from the vent with oxygen from the water to power the production of food. The worm's upper end is a deep-red, fan-like plume that absorbs hydrogen sulfide and oxygen for the bacteria, and carbon dioxide as raw material. Its color comes from several extraordinarily complex hemoglobins with up to 144 globin chains. These can carry oxygen, and even sulfide, without being poisoned by sulfide the way most hemoglobins are.

  Hemoglobin-like proteins turn up in bacteria, protozoans, fungi and plants, where some bind oxygen and others handle nitric oxide, carbon dioxide or sulfide compounds. Many nematode worms, molluscs and crustaceans carry very large multisubunit molecules, much larger than those in vertebrates. Earthworms and other annelids make erythrocruorin, a giant free-floating protein with a molecular mass above 3.5 million daltons. Other designs use different metals. Hemocyanin, the second most common oxygen carrier in nature, uses copper and turns blue when oxygenated. It is found in many arthropods and molluscs. Hemerythrin, an iron protein without heme, appears pink or violet when carrying oxygen. Chlorocruorin looks green when deoxygenated and red when oxygenated.

  Sea squirts add a puzzle. Their blood holds vanabins, which contain vanadium but bind little oxygen, so their function is still unknown. Myoglobin, meanwhile, gives vertebrate muscle its red or dark gray color. It is a single unit with no cooperative binding, and it stores oxygen rather than moving it. In alfalfa and soybeans, leghemoglobin shields the nitrogen-fixing bacteria in the roots. The enzyme nitrogenase cannot reduce nitrogen gas in the presence of free oxygen.

  Hemoglobin even works inside the brain. The A9 dopaminergic neurons of the substantia nigra, along with astrocytes and mature oligodendrocytes, contain it. It may store oxygen for these energy-hungry neurons, which also face heavy oxidative stress. That vulnerability may help explain why they degenerate in Parkinson's disease. The dark color that gives the substantia nigra its name comes from neuromelanin, not from hemoglobin-derived iron.

  The ancients linked the orange-red planet Mars to the Roman god of war, and the color of blood to rust. A common misconception holds that iron gives blood its red color. In fact the color comes from the porphyrin ring to which the iron is bound. In 2005, the artist Julian Voss-Andreae built Heart of Steel (Hemoglobin) from glass and weathering steel, shaped after the protein's backbone. The steel was meant to rust, echoing the binding of oxygen to iron. In Montreal, Nicolas Baier made Lustre (Hémoglobine), a stainless steel sculpture of the molecule about 10 meters on each side, set in the atrium of a McGill University Health Centre research building.

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• 8. Reading the Blood

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  For men, normal hemoglobin runs from 13.8 to 18.0 grams per deciliter, while for women it runs from 12.1 to 15.1. Children fall between 11 and 16, and pregnant women between 11 and 14. The test is one of the most common in medicine, usually part of a complete blood count, and it is routinely run before or after blood donation. Hematocrit, the share of blood volume taken by red cells, is typically about three times the hemoglobin figure. A reading of 17 grams per deciliter goes with a hematocrit of 51%. Dehydration or overhydration can distort the numbers.

  A concentration below normal is anemia, and doctors sort anemias by the size of the red cells: microcytic if small, macrocytic if large, normocytic otherwise. In the Western world, iron deficiency is the most common cause. Without iron, heme synthesis falls, and red cells turn hypochromic, lacking red pigment, and microcytic. Blood loss, poor nutrition, bone marrow problems, chemotherapy and kidney failure can also lower hemoglobin. Too much hemoglobin, called polycythemia, can follow high altitude, smoking, dehydration, advanced lung disease and certain tumors.

  Glucose slowly and spontaneously attaches to the terminal valine of each beta chain, forming hemoglobin A1c. Because red cells live roughly 120 days, the A1c percentage reflects average blood sugar over that stretch, which saves doctors from taking many samples. The normal range is about 4.0-5.9%. For people with type 2 diabetes, values under 7% are recommended, above 9% signals poor control, and above 12% very poor control. Heavy glycation also raises hemoglobin's grip on oxygen, which in extreme cases starves tissues.

  Light reveals oxygen levels. Oxyhemoglobin and deoxyhemoglobin absorb light differently at 660 and 940 nanometers, a difference that a pulse oximeter uses to read oxygen in a patient's blood. The same contrast explains cyanosis, the blue to purplish tint of tissues short of oxygen. Near-infrared spectroscopy tracks the two forms continuously and without needles, in work ranging from elite sports training to neonatal research. Functional magnetic resonance imaging relies on deoxyhemoglobin, which is weakly attracted to magnetic fields, while oxygenated hemoglobin is weakly repelled.

  One method for measuring total hemoglobin mass has been used for more than 100 years. A person inhales a small volume of pure carbon monoxide and rebreathes it for a few minutes. The gas binds to hemoglobin in the red cells. From the rise in carbon monoxide in the blood, the dilution principle yields the total mass of hemoglobin.

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