Williams Hematology



Structure of Hemoglobin

Primary Structure of Globin Chains

Secondary Structure

Tertiary Structure of a and b Chains

Quaternary Structure

Binding of 2,3-Bpg To Deoxyhemoglobin Tetramer

Structural Changes that Occur on Ligand Binding

The Structural Basis of Oxygen Affinity In T and R States and the Bohr Effect

The Two-State Model
Oxygen Equilibria of Hemoglobin-the Oxygen Dissociation Curve

Oxygen Affinity of Hemoglobin

Factors that Affect Oxygen Affinity

Effects of 2,3-Bpg on Oxygen Equilibria

Cooperative Interactions

The Hill Plot
Kinetics of Reactions with Ligands
Ligands of Hemoglobin

Methemoglobin and Sulfhemoglobin


Nitrosohemoglobin and Nitrosyl Heme Derivatives
Embryonic and Fetal Hemoglobins
Posttranslational Modifications of Hemoglobin
Chapter References

As a gas transport protein hemoglobin has remarkable properties, reflecting structural changes in tetrameric deoxyhemoglobin as its heme groups bind four oxygen molecules. Among the structural changes are changes in position of the heme iron and in the intersubunit hydrophobic and salt bonds of deoxyhemoglobin that are broken as ligands bind to hemoglobin. Liganded hemoglobins that do not transport oxygen, present in trace amounts normally, include methemoglobin, in which the heme iron is in the ferric (oxidized) form; carboxyhemoglobin, in which carbon monoxide is bound to the heme iron and dissociates much more slowly than oxygen; and nitrosohemoglobin, in which nitric oxide is bound to heme iron and dissociates even more slowly than carbon monoxide. Increases in methemoglobin or carboxyhemoglobin may result from toxic exposures. The many physiologic functions of nitric oxide (endothelial relaxing factor) have focused attention on the high affinity of the heme groups for nitric oxide and the potential oxygenation-linked binding of nitric oxide by the b93 cysteine residues.

Acronyms and abbreviations that appear in this chapter include: BPG, bisphosphoglycerate; cGMP, cyclic guanosine monophosphate; Hb, hemoglobin; HbL, liganded hemoglobin.

Hemoglobin functions to carry oxygen from the lungs to the tissues and transport carbon dioxide from the tissues to the lungs, and it serves also to destroy the physiologically important nitric oxide molecule. It has evolved to perform its transport functions in a highly efficient manner: (1) The oxygen affinity of hemoglobin permits nearly complete saturation with oxygen in the lungs, as well as efficient oxygen unloading in the tissues; (2) Its affinity increases with oxygenation, resulting in the sigmoid shape of the oxygen dissociation curve; and (3) deoxyhemoglobin binds protons and oxyhemoglobin releases protons. The last property, expressed as the alkaline Bohr effect, also facilitates oxygen loading in the lungs and unloading in the tissues. The Perutz models of oxygenated and deoxygenated hemoglobin provide important insights into the structural basis of these three major features of the equilibria of oxygen with hemoglobin. The reader is referred to two1,2 of the many excellent sources for a detailed analysis of structure-function relationships.
The roles of different parts of the hemoglobin molecule in its equilibria have been deduced from its amino acid sequence, its helical conformation, models derived from x-ray crystallography,3,4 studies of the kinetics of reactions of hemoglobin with ligands,5 and observations utilizing nuclear magnetic resonance.6 The concentration of hemoglobin within human red cells is extraordinarily high (34 g/dl), and its efficiency as an oxygen carrier is enhanced by its packaging in flexible cells of optimal shape for the diffusion of gases.
Normal mammalian hemoglobins contain two pairs of unlike polypeptide chains: one chain of each pair is a or a-like and the other is non-a (b, g, or d). The a chains of all human hemoglobins encountered after early embryogenesis are the same. The non-a chains include the b chain of normal adult hemoglobin [hemoglobin A (a2b2)], the g chain of fetal hemoglobin [hemoglobin F (a2g2)], and the d chain of hemoglobin A2 [hemoglobin A2 (a2d2)], the minor component which accounts for 2.5 percent of the hemoglobin of normal adults.
In the amino acid sequence of each polypeptide chain, certain residues appear to be critical to stability and function. Such residues are usually the same (invariant) in a or b chains. The NH2-terminal valines of the b chains are important in 2,3-BPG interactions (bisphosphoglycerate has replaced the older term diphosphoglycerate). The C-terminal residues are important in the salt bridges that characterize the unliganded molecules. Areas of contact between chains and between heme and globin tend to contain invariant residues. Unlike many proteins, native hemoglobin contains no disulfide bonds: of its six -SH groups (cysteine residues a104, b93, and b112), only the two b93 residues are exposed to the solvent.
The non-a (b, g, d or e) chains are all 146 amino acids in length; the b chain begins with valine and histidine. The C-terminal residues are Tyr b145 and His b146. The d chain (of hemoglobin A2) differs from the b chain (of hemoglobin A) in only 10 residues. The first eight residues and the C-terminal residues (127 to 146) are the same in d and b chains. Tetramers of b chains (hemoglobin H) may be found in a thalassemia.
The g chain of fetal hemoglobin (hemoglobin F) differs from the b chain by 39 residues. The N-terminal residues of the g chain and b chain are glycine and valine respectively, while the C-terminal residues, Tyr145 and His146, are the same as in g and b chains. Appreciable quantities of free g chains are found in the red cells of some infants with a thalassemia; free g chains, like b chains, can form homotetramers known as hemoglobin Bart’s. In addition to the different N-terminal residues, several other differences in primary structure between the g and b chains are noteworthy: the g chain contains isoleucine, while the b chains do not. The increased alkali resistance of hemoglobin F and hemoglobin Bart’s (g4) has been attributed to the different amino acids at residues 112 and 130 (bCys and Tyr by gThr and Trp, respectively).
The g genes are duplicated: one codes for glycine (Gg) and the other for alanine (Ag)7 at residue 176, giving rise to two kinds of g chains. In addition, a common polymorphism, the substitution of threonine for isoleucine, is frequently found at residue 75 of the Ag chain.
About 75 percent of the amino acids in a or b chains are in a helical arrangement. All studied hemoglobins have a similar helical content (Fig. 28-1a). Eight helical areas, lettered A to H, occur in the b chains. Hemoglobin nomenclature specifies that amino acids within helices are designated by the amino acid number and the helix letter, while amino acids between helices bear the number of the amino acid and the letters of the two helices. Thus, residue EF3 is the third residue of the segment connecting the E and F helices, while residue F8 is the eighth residue of the F helix. Alignment according to helical designation makes homology evident: residue F8 is the proximal heme-linked histidine, and the histidine on the distal side of the heme is E7.

FIGURE 28-1 (a) The representation of the structure of b chains. Arrows indicate sites of substitutions in a number of unstable hemoglobins. (b) The hemoglobin molecule, as deduced from x-ray diffraction studies, shown from above. The molecule is composed of four subunits: two identical a chains (light blocks) and two identical b chains (dark blocks). 2,3 BPG binds to the two b chains in the deoxyhemoglobin molecule. (c) Schematic diagram of rotation of a2b2 dimer relative to a1b1 in quaternary structure change from deoxyhemoglobin (solid lines) to carboxyhemoglobin (dashed lines). Modified slightly from J Mol Biol 129, J. Baldwin and C. Chothia, Haemoglobin: the structural changes related to ligand binding and its allosteric mechanism, page 196, 1979, by permission of authors and publisher, Academic Press Ltd., London.

The tertiary structure of the a and b chains is shown in Fig. 28-1b. The prosthetic group of hemoglobin is ferroprotoporphyrin IX. Its structure is shown in Fig. 28-2a. The heme group is located in a crevice between the E and F helices in each chain (Fig. 28-2b). The highly polar propionate side chains of the heme are on the surface of the molecule and are ionized at physiologic pH. The rest of the heme is inside the molecule, surrounded by nonpolar residues except for two histidines. The iron atom is linked by a coordinate bond to the imidazole nitrogen (N) of histidine F8; the E7 distal histidine, on the other side of the heme plane, is not bonded to the iron atom but is very close to the ligand-binding site.

FIGURE 28-2 (a) Structure of heme (ferroprotoporphyrin IX). (b) Heme group and its environment in the unliganded a chain. Only selected side chains are shown: the heme 4-propionate is omitted (Gelin, et al8).

In both types of chains there is a preponderance of nonpolar residues in the immediate vicinity of heme. On the proximal side, the important residues in contact with heme are Val FG5, Leu FG3, His F8, Leu F7, Leu H19, and Leu F4; on the distal side, important heme contacts are Phe CD4, His E7, Leu G8, Val E11, and Lys E10. The V produced by helices E and F provides the main walls of the heme pocket. Helices B, G, and H form the floor, and the segments C and CD guard the opening to this pocket. Important features of the heme pocket are as follows:

The hydrophobic “cage” around the heme provides the main stabilizing force for the binding of heme to the protein. The closely packed side chains that constitute the cage do not allow significant movement of the heme.

In a nonpolar environment it is much more difficult to oxidize Fe2+ to Fe3+. This feature facilitates binding of oxygen without oxidation.

In b subunits of deoxyhemoglobin the methyl group of Val E11 is in van der Waals’ contact with porphyrin. It overlaps the van der Waals radii of heme ligand(s) (O2, CO, and NO). This steric hindrance by Val E11 is much smaller in a chains.4

Two atoms of the distal histidine (E7) are in van der Waals’ contact with the porphyrin in both oxyhemoglobin and deoxyhemoglobin, and the imidazole N also overlaps with the ligand-binding site. The histidine side chain also acts as a gate to the ligand-binding site, not allowing ligand to enter or leave unless it swings out of the way.

The hydrophobic residues in the C and CD segments that guard the opening to the heme pocket effectively exclude polar ligands from entering the heme pocket.

The imidazole N of the proximal histidine is hydrogen-bonded to the carboxyl of Leu F4 and the OH of Ser F5. This hydrogen bonding and the neighboring side chains hold the proximal His F8 in position rather rigidly.

Both a and b hemes form about 75 contacts with 30 atoms in 16 globin residues in each heme pocket.
Although the x-ray structure of deoxyhemoglobin indicates a planar porphyrin with iron displaced 0.6 Å out of the plane in a chains and 0.63 Å in b chains, data on model compounds and considerations based on energy-minimized geometry suggest that the plane of pyrrole nitrogens might be displaced toward iron by 0.22 Å in a chains and by 0.17 Å in b chains compared with the mean plane of the porphyrin carbons. The energy difference between the domed and the planar porphyrin structure is small. The iron atom in both deoxyhemoglobins and methemoglobins is high-spin, and it has been suggested that its ionic radius is too large to fit into the plane of the porphyrin ring. Spin state of hemoglobin derivatives depends on the number of unpaired electrons in d orbitals of iron. In both subunits of deoxyhemoglobin, the imidazole ring of the proximal His F8 is in an asymmetric position with respect to the porphyrin nitrogens of the heme, such that the atom Ce is closer to porphyrin N(1) than Cd is to N(3)8 (see Fig. 28-2b).
In both the a and the b subunits of human deoxyhemoglobin, the ligand-binding site is blocked. In the a subunits a water molecule is attached to the distal His E7. There is no direct bond between the water molecule and the iron atom. In b chains, the methyl group of Val E11 lies within 1.8 Å of the ligand-binding site. Both of these groups would have to move out of the way before the ligand could bind to iron.4
In the deoxy state, the hemoglobin tetramer is held together by intersubunit salt bonds and intersubunit hydrophobic contacts, in addition to a certain number of hydrogen bonds.

The intersubunit salt bonds (Fig. 28-3). Four of these salt bonds, involving Arg HC3(141), are between the two a chains. The two salt bonds involving His HC3(146) are between b and a chains. There are two intramolecular salt bonds in b chains.

FIGURE 28-3 Salt bridges in deoxyhemoglobin (*= ionizable group less protonated at pH 9.0 than at pH 7.0). These groups account for 60 percent of the alkaline Bohr effect. The remainder is due to His aH5 (Perutz2).

The a1b1 (or a2b2) and a1b2 (or a2b1) intersubunit contacts (see Fig. 28-1b).

The a1b1 contact. This more extensive contact, between a1 and b1 subunits, involves 32 residues, including 126 atoms, 4 hydrogen bonds, and 1 solvent-mediated hydrogen bond.

The a1b2 contact. Slightly less extensive than the a1b1 contacts, this contact involves 27 residues, including 107 atoms, 6 hydrogen bonds, and 3 solvent-mediated hydrogen bonds.
In deoxyhemoglobin, 2,3-BPG is situated in the central cavity between the two b chains (see Fig. 28-1b).9 The phosphate groups form salt bonds with b N-terminal amino groups and the imidazoles of b2 and b143 histidine; the carboxyl groups bind to Lys b82.
The changes that occur on going from the deoxy to the oxy structure (Fig. 28-1c) are of two types: the tertiary structural changes within the subunits of a1b1 (or a2b2) dimer, and a quaternary structural change in which the position of a1b1 changes relative to a2b2. The two structural changes are linked.
Iron-to-ligand bond formation would require moving the iron atom toward the heme plane. This would bring the Ce atom of the proximal histidine too close to both porphyrin N(1) and C atoms in the pyrrole ring, producing large steric strain. The tertiary structural changes involving heme, proximal histidine, F helix, FG corner, and others all minimize the steric strain produced as a result of metal-to-ligand bond formation.
The most important of the tertiary structural changes seems to be the translation of the F helix approximately 1 Å across the heme plane, the tilting of the F helix with respect to the heme, and the movement of heme and the FG corner toward the center of the molecule. This movement of the F helix takes the proximal histidine from its asymmetric position in deoxyhemoglobin to a more symmetric position in liganded hemoglobin. The motion of b hemes removes the ligand-binding site from the vicinity of Val bE11, which may hinder ligand binding in the deoxy state. These changes in the tertiary structure are linked to the quaternary changes through the motion of FG corners. The C helices and FG corners of the a1b1 dimer are in contact with the FG corners and C helices of a2b2 in both quaternary structures. The contacts between a1FG and b2C (and a2FG and b1C) act as “flexible joints” and undergo only small relative motions. The contacts between a1C and b2FG (and a2C and b1FG) act as switch regions that have two different stable positions. The change between the two stable positions involves a relative movement of approximately 6 Å.
The quaternary structural change that occurs on ligand binding to hemoglobin involves rotation of the a2b2 dimer interface relative to the a1b1 by 14.9° and translation of 0.8 Å. The overall number of intersubunit hydrogen bonds and contacts may not change significantly, but they become less stringent.
The carboxy salt bridges involving Arg aHC3(141) are not made in the quaternary liganded structure because the space between a1b1 and a2b2 is too narrow for these residues to occupy the position they have in the deoxy quaternary state. The carboxy-terminal residues His bHC3(146) are separated from Lys aC5(40), the group to which they are bonded in the deoxy state, by a 7 Å shift that occurs in these regions in the quaternary structural change.
The rotation and translation of the a2b2 dimer with respect to the a1b1 dimer, mentioned earlier, also renders the central cavity too small, especially the gap between the H helices of the b chains, for the binding of 2,3-BPG to the two b chains. In addition, the distance between the a-amino groups increases from 16 to 20 Å, so these groups cannot bind to the phosphates of 2,3-BPG. All these structural changes cause expulsion of 2,3-BPG from the hemoglobin tetramer in the fully liganded hemoglobin (see Fig. 28-1b). The quaternary structures of unliganded and liganded hemoglobin are known as the T-state (“tense”) and R-state (“relaxed”) structures, respectively. Respective alternative terms are low-affinity and high-affinity states or deoxy and oxy states (Table 28-1).


One mechanism proposed to account for the differing oxygen affinities of the T and R states and for the Bohr effect11 takes into account the x-ray crystallographic data and calculations of energies associated with various structural changes that might occur at the heme on ligand binding.8,12,13 The low affinity of deoxyhemoglobin for its first ligand appears to be due to the strain induced by the steric repulsion arising from the position of the F helix and, in particular, from the position of the proximal histidine relative to the heme; the imidazole ring of each chain is tilted so that its interaction with porphyrin-ring carbons provides steric hindrance in iron-to-oxygen bond formation. Quaternary structural changes brought about by the tertiary structural changes via the changes at the intersubunit contacts (particularly at b2FG-a1C and b1FG-a2C) place the proximal histidine in R structure in a symmetric position with respect to the plane of the porphyrin ring and thus minimize the steric interaction between the imidazole (Ce) and porphyrin N(1) and a carbon atom in the pyrrole-1 ring. In b subunits, an additional factor increases ligand affinities in the R state: the translational and rotational movements of b hemes remove the ligand-binding site from the vicinity of the Val E11 side chain. Both of these factors result in a much stronger iron-to-oxygen bond in R structure.
Calculations8,13 suggest that the geometry of the heme in deoxyhemoglobin may be very similar to that in isolated heme. If this is correct, then the iron atom in deoxyhemoglobin is in its optimal position for five-coordinate high-spin Fe2+ ion, and there is little strain on the unliganded heme. Instead, the heme in the liganded subunit in the deoxy quaternary tetramer would be under strain.
The structural change described above also results in an altered electrostatic environment of certain protonated amino acid side chains (see Fig. 28-3), and their proton dissociation constants are increased:

This equation forms the basis of the Bohr effect, discussed under “Oxygen Equilibria of Hemoglobin—the Oxygen Dissociation Curve,” below.
The mechanisms of cooperative ligand binding by hemoglobin proposed by various workers must be regarded as tentative, since none of them is capable of explaining all the physicochemical properties of hemoglobin even qualitatively. It is not clear in these models at which point in ligation the transition from low-affinity (T) to high-affinity (R) structure takes place. In the most widely accepted model,14 T and R species are considered to be in rapid equilibrium:

and KR and KT are the ligand dissociation constants in the R and T states respectively. A shift in the equilibrium between T and R species with fractional saturation would then account for the cooperative ligand binding and other features of the oxygen dissociation curve. The transition point from T to R structure acquires a statistical meaning and is given by the equation

where nt is the number of ligands bound at the switch-over point from T to R. Depending on the experimental conditions, the value of nt for hemoglobin lies in the range of 2.3 to 3.0.
Important gas-transport properties of hemoglobin are evident from inspection of the oxygen dissociation curve (Fig. 28-4). The oxygen affinity increases with increasing oxygen saturation of the hemoglobin. This increasing oxygen affinity with increasing saturation is described by the sigmoid shape of the oxygen dissociation curve. The terms heme-heme interaction and cooperative interactions also describe this change in oxygen affinity as a function of saturation. The binding of more protons by deoxyhemoglobin than by oxyhemoglobin, known as the Bohr effect, is reflected in the left shift of oxygen dissociation curves with increasing pH. These functional properties are interdependent, or linked.14 The oxygen affinity depends on the state of oxygenation (cooperative interactions) and on the pH (Bohr effect).

FIGURE 28-4 Oxygen dissociation curve of human hemoglobin. Inserts: Effect of temperature (upper) and Bohr effect (lower) (Comroe15).

For clinical usage, the oxygen affinity of hemoglobin is usually expressed in terms of the P50, the oxygen tension at which hemoglobin is half saturated. This value is 26 torr in normal red cells or concentrated hemolysates at 37°C and plasma pH of 7.4. (The pH of the interior of the red cell is about 0.2 units lower than the pH of the plasma.) The partial pressure of oxygen in room air is about 100 torr; in the pulmonary alveoli it is about 95 torr. Oxygen diffuses passively across the alveolar capillary membrane during the time (<1 s) the red cell spends within the pulmonary vasculature. Desaturated blood from the bronchial (and other) veins returns to pulmonary veins, resulting in a Po2 of about 90 torr in the left side of the heart; i.e., systemic arterial blood is almost fully saturated with oxygen. As the blood traverses the systemic capillaries, the release of oxygen is determined by the Po2 of the tissues. The steep portion of the oxygen dissociation curve allows a relatively large amount of oxygen to be unloaded for a small decrement in Po2 The Po2 in the capillaries of different organs varies with oxygen consumption.
The value of P50 is taken from the midpoint of the oxygen dissociation curve and does not reflect the shape of the curve. With increasing oxygen affinity, the value for P50 becomes smaller; i.e., the dissociation curve is “shifted to the left.” High values for P50 indicate lower oxygen affinity of the hemoglobin. Since hemoglobin is nearly fully saturated with oxygen at a Po2 of 85 torr, a right shift in the dissociation curve will facilitate oxygen delivery, and nearly full saturation will still occur in the lungs.
The three primary determinants of the value of P50 are temperature, pH, and red cell 2,3-BPG concentration. The increase in oxygen affinity with lower temperatures is observed in red cells or in hemoglobin solutions.
The Bohr effect is observed in red cells and hemoglobin solutions (see inset of Fig. 28-4). With increasing hydrogen ion concentration (decreasing pH), P50 increases; i.e., oxygen affinity declines. Protons stabilize the T state by stabilizing the intersubunit bonds and the bonds between the two b chains and 2,3-BPG; 2,3-BPG and other anions stabilize the T state by complexing preferentially with hemoglobin in this state. Both these factors shift T-to-R equilibrium toward the T (low-affinity) state.
The Bohr shift constitutes an important buffer system of the body. When blood reaches the tissues, where the oxygen tension is lower and the hydrogen ion concentration is increased by lactic acid or carbon dioxide, the Bohr shift of the dissociation curve makes more oxygen available. As the hemoglobin loses its oxygen and the unliganded form binds protons, changes in hydrogen ion concentration are minimized. The proton binding of deoxyhemoglobin provides an important part of carbon dioxide transport: Carbon dioxide diffuses into the red cell, and its conversion there to bicarbonate is catalyzed by carbonic anhydrase. The bicarbonate ion leaves the red cell, and the hydrogen ion is bound by deoxyhemoglobin. The ultimate effectiveness of this physiologic buffer system depends on the ease with which carbon dioxide or bicarbonate is retained or eliminated in the lungs and kidneys. The Bohr effect is also observed in the reactions of hemoglobin with ligands other than oxygen (e.g., carbon monoxide, ethyl isocyanide) and in the oxidation of hemoglobin to methemoglobin (the oxidation Bohr effect).
Carbon dioxide reacts with N-terminal residues of the b chains of hemoglobin to yield carbamino derivatives, a phenomenon separate from the Bohr effect. These carbamino derivatives are of minor importance in carbon dioxide transport by hemoglobins that bind organic phosphates.
The usual P50 measurements are done under standard conditions of temperature, pH, and Po2 Therefore, observed variations in the oxygen affinity are usually related to the concentration of 2,3-BPG16 (or less commonly to the presence of structurally different hemoglobins with altered oxygen affinity). The P50 of whole blood or of hemoglobin solutions increases with increasing concentrations of 2,3-BPG (the normal value of 2,3-BPG is about 5 mmol/liter of packed red cells). The relationship of 2,3-BPG and P50 is not linear: with higher concentrations, smaller increments in P50 are observed. In addition to its stabilization of the deoxy form of the tetramer, 2,3-BPG, because it is a nonpermeating anion, lowers intracellular pH relative to plasma pH. Other factors, such as carbon dioxide in carbamino linkage and mean corpuscular hemoglobin concentration, probably do not much affect the oxygen affinity of normal hemoglobin under conditions encountered clinically, although the carbon dioxide effect is rather large when the P50 values at pH 7.2 are compared at Po2 values ranging from 0 to 40 torr. A concentration-dependent decrease of oxygen affinity in sickle hemoglobin reflects the gelation of deoxygenated hemoglobin S.
Increases in P50 may be observed in acidosis or in any state (anemia, hypoxia, ascent to high altitudes) in which 2,3-BPG is increased. A higher oxygen affinity (lower P50) is seen in hemoglobins modified by treatment with a number of agents, including cyanate. The role of organic phosphates in regulating oxygen delivery to the tissues has provided an explanation for the finding that although the oxygen affinity of cord blood exceeds that of maternal blood, the oxygen affinity of isolated hemoglobin F does not differ greatly from that of hemoglobin A. The oxygen affinity of phosphate-free hemoglobin F is lower than that of hemoglobin A, but the effect of 2,3-BPG on the oxygen affinity of hemoglobin F is much less than that on adult hemoglobin.1 The oxygen affinity of fetal blood is therefore higher than that of adult blood, permitting more complete placental extraction of oxygen from maternal blood.
The physiologic advantages that derive from the sigmoid shape of the oxygen dissociation curve are obvious from the data in Fig. 28-4. A fall in Po2 from 100 to 60 torr results in a decline in oxygen saturation from 97.5 to 89 percent, while a fall from 60 to 20 torr will be accompanied by a decline in oxygen saturation from 89 to 35 percent and the release of more than 10 ml oxygen per deciliter of blood to the tissues. The rather “flat” portion of the oxygen dissociation curve at Po2 from 70 to 100 torr results in nearly complete saturation of hemoglobin even at the lower partial pressures of oxygen found at quite high altitudes. The advantages of decreased oxygen affinity as a compensatory mechanism in hypoxemia obtain only if the sigmoid shape of the dissociation curve is preserved.
The plot of log [y /(1 – y)] against log Po2 is known as the Hill plot. The Hill equation is

The n value (the slope from the Hill plot at half saturation) is taken as a convenient measure of cooperativity. Values of n in noninteracting hemoglobins which exhibit hyperbolic oxygen dissociation curves (e.g., myoglobin and hemoglobin H) are about 1. In a normal tetrameric hemoglobin with four oxygen-reactive sites, the maximum value for n would be 4.0; however, n values of 2.7 to 3.0 rather than 4.0 are encountered in normal hemoglobin.
The reactions of hemoglobin with ligands are much faster than would be needed for reaction during transit in the microvasculature. The exception to this is the slow rate of dissociation of carbon monoxide and nitric oxide from hemoglobin, which results in the extremely high affinity of these ligands for hemoglobin and prevents equilibration of carbon monoxide- or nitric oxide-containing red cells with oxygen in the lungs. The oxygen dissociation curve does not reveal certain finer details of the overall reaction between hemoglobin and ligands (i.e., O2, CO, or NO) that can be observed when one considers the rates of ligand binding (the “on” rates) and the rates of ligand dissociation (the “off” rates) separately:

The stepwise binding of ligands to hemoglobin tetramer can be written in terms of the Adair four-step model.17 Eight Adair rate constants, four stepwise “on” and four “off” for each ligand (O2, CO, NO), and the ligand-specific equilibrium constants have been determined.18,19 and 20 However, because of the cooperative nature of the reactions, accurate determination of the intermediate equilibrium and rate constants (steps 2 and 3) is not easy, and constants for those two steps are less certain.
The “on” reactions of oxygen, carbon monoxide, and nitric oxide with deoxyhemoglobin are strictly first-order in ligand and hemoglobin concentrations; hence, the overall reaction is second-order. The reaction rates accelerate as the reaction proceeds. The initial rates approximate the rate constant for the formation of the monoliganded species, Hb4L.5 Oxyhemoglobin and particularly carboxyhemoglobin are photosensitive, and each loses its ligand on exposure to light. This property has been used to study the rate constants for the formation of the fully liganded species. Five percent or less of ligand is removed from fully liganded carboxyhemoglobin using a brief (e.g., 2 µs to 1.5 ms) flash of strong light; rates of subsequent ligand recombination with the triliganded species are observed.21 This reaction is 40 times as rapid as that of ordinary deoxyhemoglobin. The quickly reacting unliganded hemoglobin (designated Hb*) is thought to be unliganded hemoglobin trapped in the high-affinity conformation. It has been estimated that Hb* decays into the regular deoxy conformation by a first-order process.
The ligand dissociation rates from oxyhemoglobin, Hb4(O2)4, and nitrosohemoglobin, Hb4(NO)4, also accelerate as the reaction proceeds and are strongly affected by phosphates. For carboxyhemoglobin, the ligand dissociation reaction rates are not as much affected by phosphates.
The kinetic studies bring out certain interesting features of ligand binding to hemoglobin that are not apparent from the ligand dissociation curve alone:

The cooperativity in the reactions of hemoglobin, if considered in terms of “off#148; and “on” rates, is highly dependent on the nature of the ligand. Thus, while carbon monoxide shows cooperativity mainly in the ligand combination rates, oxygen and nitric oxide show cooperativity in the ligand dissociation rates. These differences arise from the differences in the stereochemistry of these ligands. Oxygen prefers bent bonding (with respect to the axis perpendicular to the heme plane) with the iron atom in heme and avoids steric interaction with the residues on the distal side of heme. The variation in “off” rate constants in this case mainly reflects the tension on the Fe-O2 bond owing to steric and/or electronic factors originating on the proximal side of heme. This should also be true for nitric oxide. Carbon monoxide, however, prefers linear bonding with the iron atom in heme. The residues on the distal side of heme in the heme pocket—particularly His E7 and Val E11 in b chains—are situated too close to the ligand-binding site and therefore provide steric hindrance in the formation of the iron-to-ligand bond. Variations in the stepwise “on” rate constants for carbon monoxide therefore may reflect the variations in the steric hindrance from the distal residues, in addition to the proximal side effects discussed above. From the values of carbon monoxide “off” rates it is also obvious that the Fe-CO bond is too strong to respond to the same extent as the Fe-O2 bond to the “tension” from the proximal side. It has been suggested that the heme pocket discriminates between these three ligands by electrostatic interactions with the bound ligand. Favorable electrostatic interactions stabilize the bound oxygen by a factor of about 100, nitric oxide by about 10, and carbon monoxide only by 2–3.

The effect of phosphates in oxyhemoglobin is due primarily to changes in the “off” rates rather than to changes in the “on” rates; species in the quaternary T state show larger enhancement in the dissociation rates.18 Various equilibrium and kinetic studies suggest that although phosphates bind to b chains, they affect the properties of a chains more.

Kinetic studies indicate that a and b chains in hemoglobin tetramers have different reactivity. These differences are shown in the reactions of deoxyhemoglobin as well as in fully liganded hemoglobin.5 The magnitude of these differences is smaller in the reactions of ferrohemoglobin than in those of ferrihemoglobin.

Nitric oxide binds to hemoglobin 105 to 107 times more strongly than do carbon monoxide and oxygen. The half-life for the disappearance of nitrosohemoglobin is approximately 11 h. Ligand combination rates of carbon monoxide are activation controlled; i.e., the protein has to undergo some structural change before carbon monoxide can form a covalent bond with heme. These rates therefore depend on the protein structure. The combination rates of oxygen and nitric oxide, on the other hand, are diffusion controlled; i.e., oxygen-to-heme and nitric oxide-to-hemebond formation take place prior to any major change in protein structure. Therefore, the reaction rates of oxygen and nitric oxide depend much less on protein structure than do the rates for carbon monoxide.
Methemoglobin is hemoglobin in which the iron has been oxidized (a23+b23+). This oxidized hemoglobin is no longer capable of reversibly binding oxygen. Methemoglobin is reddish brown, with an absorbance peak at 630 nm at acid pH (Fig. 28-5). The absorption spectrum of methemoglobin is strongly pH-dependent: at low pH, a water molecule is bound to the iron and occupies the space between the ferric iron and the distal histidine. At alkaline pH, hydroxyl ion is bound to the ferric iron. At an acid pH the iron atom of aquomethemoglobin is in the high-spin state and goes to a low-spin state with increasing pH. In a mixture of methemoglobin and normal ferrous hemoglobin, intermediates called valency hybrids are formed. These are tetramers in which varying numbers of hemes are in the ferrous state and the remainder are in the ferric state. These valency hybrids have been useful models for studying subunit cooperativity. In a hemoglobin solution containing oxy, deoxy, and methemoglobin species the oxygen affinity is unusual; i.e., the dissociation curve is left-shifted and P50 is decreased. This increase in oxygen affinity provides the explanation for the toxicity of methemoglobinemia: in addition to the nonfunctional ferric subunits, the increased oxygen affinity of ferrous hemes accompanying ferric hemes in tetramers impairs oxygen delivery. Methemoglobin solutions are believed to be equilibrium mixtures of quaternary R+T structures. Ingestion of certain drugs or accidental ingestion of oxidizing agents may lead to methemoglobinemia (see Chap. 49).

FIGURE 28-5 Spectra of some hemoglobin derivatives. Extinction coefficients plotted against wavelength (pH 7.4).

On exposure to some toxic agents, sulfhemoglobin is formed. Sulfhemoglobin is a curious compound in which the iron is in the ferrous state but the oxygen affinity is about 100 times lower than that of normal hemoglobin. The sulfur is not liganded to the iron but is found in the porphyrin ring (see Chap. 49).
Carboxyhemoglobin (carbonmonoxyhemoglobin) results from the binding of carbon monoxide to the heme iron. In carboxyhemoglobin the carbon monoxide is bound at an angle different from that of oxygen relative to the plane of the heme. The rate of association of carbon monoxide with hemoglobin is slower than that of oxygen; however, carbon monoxide dissociates much more slowly than does oxygen. Hemoglobin binds carbon monoxide about 200 times more strongly than it binds oxygen. If the partial pressure of carbon monoxide is 0.5 percent that of oxygen, the concentration of carboxyhemoglobin at equilibrium will approximately equal that of oxyhemoglobin. The symptoms of carbon monoxide poisoning result from tissue oxygen deprivation. Hemoglobin tetramers, in which some hemes are bound to oxygen and others to carbon monoxide, have high oxygen affinity. Carbon monoxide, like oxygen, binds reversibly to the heme iron of hemoglobin. However because of its slow dissociation from the heme iron, the affinity of carbon monoxide for hemoglobin is approximately 210-fold higher than the affinity of oxygen. Therefore, inhalation of carbon monoxide even at low partial pressures leads to significant levels of carboxyhemoglobin in the blood. For example, a concentration of 0.1% carbon monoxide in inspired air will result at equilibrium in 50% carboxyhemoglobin. Oxygen transport and delivery are impaired both by the unavailability of carboxyhemoglobin for oxygen transport and by the higher oxygen affinity of the mixed oxygen/carbon monoxide tetramers. In addition, carbon monoxide poisoning leads to various neurologic disorders, sometimes occurring several days after exposure. Mechanisms leading to neurologic deficits are not understood, but increased levels of reactive oxygen species, including peroxynitrite, and/or impairment of electron transfer have been suggested. At carboxyhemoglobin blood levels greater than 30–40%, dizziness, headache, and loss of consciousness occur; concentrations of carboxyhemoglobin greater than 50%–60% are usually lethal. There is no direct correlation between levels of carboxyhemoglobin and development of neurologic dysfunction; duration of unconsciousness has better correlation with outcome. The half-life of carboxyhemoglobin in vivo can be reduced by 50 percent by the administration of oxygen. In severe poisoning, the use of hyperbaric oxygen if available will reduce the levels of carboxyhemoglobin even more rapidly.
Nitric oxide, another physiologic ligand of hemoglobin, has attracted interest as the production of nitric oxide and its different physiologic functions have been demonstrated in a variety of tissues.23 Nitric oxide stimulates a cytosolic heme protein, guanylate cyclase, which catalyzes the formation of cGMP, a mediator of many biological reactions, including vascular smooth muscle relaxation, inhibition of platelet aggregation, and macrophage cytotoxicity. The activity of guanylate cyclase is increased 50- to 200-fold by its reaction with nitric oxide and by exchange of nitrosyl heme from nitrosomethemoglobin and nitrosohemoglobin.23 Myoglobin and hemoglobin react with nitric oxide very rapidly and can prevent guanylate cyclase activation by nitric oxide produced endogenously. Many of the toxic effects of stroma-free hemoglobin24 have been attributed to vasoconstriction that might result from loss of endothelial-cell-produced nitric oxide by tight binding of nitric oxide to extravascular hemoglobin in the tissues. However, detailed consideration of the possible reactions of nitric oxide suggests that the explanation of toxicity may not lie in simple reactions of nitric oxide with hemoglobin.
It has been shown that in vitro carbon monoxide activates soluble guanylate cyclase by a factor of four, which is much less than the effect of nitric oxide. However, in the presence of some molecules carbon monoxide can be as potent an activator of guanylate cyclase as is nitric oxide.24 Both carbon monoxide and nitric oxide have also been shown to inhibit endothelial nitric oxide synthase. At present the physiologic significance of these in vitro observations is uncertain.
Reactions of endogenously produced nitric oxide or molecules capable of releasing nitric oxide, e.g., nitroglycerine, may result in higher levels of methemoglobin, resulting from the reaction

The reaction is irreversible and as fast as the reaction of nitric oxide with deoxy hemoglobin (Table 28-2). Nitric oxide also reacts with Cysb93 yielding a nitrosothiol of hemoglobin. Since Cysb93 is exposed to solvent in the liganded structure, its nitrosation proceeds faster in oxyhemoglobin than in deoxyhemoglobin. It has been proposed that increased binding of nitric oxide to the b93 cysteine in oxyhemoglobin and its release on deoxygenation may contribute to physiologic vasodilation in response to hypoxia.25


The rate constants for reactions of hemoglobin with oxygen, carbon monoxide, and nitric oxide are given in Table 28-2. Data for the reaction of chloride, fluoride, azide, thiocyanate, and nitric oxide with ferric hemoglobin are not included in the table. Except for CN– and F–, the reactions for these ligands are very heterogeneous, reflecting different reactivities of the a and b chains. Nitric oxide and cyanide anion bind with ferric heme more strongly than do Cl–, F–, and SCN–. In the case of nitric oxide, reaction with ferric heme is followed by fast reduction of ferric heme to ferro heme. Alkyl isocyanides and aromatic nitrosyl derivatives also react with deoxyhemoglobin, but at much slower rates than those of oxygen, carbon monoxide, and nitric oxide.
The hemoglobin of the nucleated erythrocytes of the yolk sac stage of fetal development contains two embryonic polypeptide chains, the a-like z and b-like e; these subunits decline rapidly after the eighth week of gestation, and by the tenth week, when erythropoiesis has moved to the liver, a and g chains (of hemoglobin F) account for most of the newly synthesized hemoglobin26 (Fig. 28-6). Low levels of mRNA of the q-globin gene (located 5′ to the duplicated a loci) are found in fetal and adult reticulocytes, but q hemoglobin has not been isolated.27 About the fifth week of gestation, hemoglobin Gower-1, z2e2 and hemoglobin Gower-2, a2e2, constitute 42 percent and 24 percent of the total hemoglobin, respectively, with fetal hemoglobin accounting for the remainder.1 The oxygen equilibria of the erythrocytes of the early fetus are similar to those of cord blood cells.28 As noted earlier, the higher oxygen affinity of fetal red cells reflects the smaller effect of bisphosphoglycerate on fetal hemoglobin (a2g2).

FIGURE 28-6 Changes in hemoglobin tetramers (top panel) and in globin subunits (bottom panel) during human development from embryo to early infancy. Reprinted from Hemoglobin: Molecular, Genetic and Clinical Aspects, HF Bunn and BG Forget,1 p 68 by permission of authors and publisher, WB Saunders Company.

The best known posttranslational modification of hemoglobin results from nonenzymatic glycation: a chromatographically separate component of human hemoglobin, hemoglobin A1c,29 amounts to about 3 to 4 percent of normal hemoglobin and is increased in uncontrolled diabetes mellitus.30,31,32 and 33 Hemoglobin A1c results from the nonenzymatic glycation (by glucose) of the b N-terminal valine residues, in which the initial Schiff base linkage between the bNH2 and glucose undergoes a rearrangement to form a stable ketoamine linkage.34 The observed proportions of hemoglobin A1c depend upon the concentration of glucose in the red cell during the weeks or months prior to its measurement and on the life span of the red cell.
Other posttranslational modifications of hemoglobin include acetylation of 10 to 20 percent of the g chains of hemoglobin F,35 resulting in a minor component designated hemoglobin F1, which bears a more negative charge than the main fetal component. In patients with uremia the carbamylation of the N-terminal amino group of both a and b chains of hemoglobin A is attributed to the reaction with cyanate, which is increased in renal failure.36

Bunn HF, Forget BG: Hemoglobin: Molecular, Genetic and Clinical Aspects. Saunders, Philadelphia, 1986.

Perutz MF, Wilkinson AJ, Paoli M, Dodson GG. The stereochemical mechanism of the cooperative effects in hemoglobin revisited. Annu Rev Biophys Biomol Struct 27:1, 1998.

Perutz MF, Rossman MG, Cullis AF, Muirhead H, Will G, North AGT: Structure of haemoglobin: A three-dimensional Fourier synthesis at 5.5 Å resolution, obtained by x-ray analysis. Nature 185:416, 1960.

Fermi G, Perutz MF, Shaanan B, Fourme R: The crystal structure of human deoxyhemoglobin at 1.74 Å resolution. J Mol Biol 175:159, 1984.

Parkhurst LJ: Hemoglobin and myoglobin ligand kinetics. Annu Rev Phys Chem 30:503, 1979.

Ho C: Proton nuclear magnetic resonance studies on hemoglobin: cooperative interactions and partially ligated species. Adv Protein Chem. 43:153, 1992.

Schroeder WA, Huisman THJ, Shelton JR, et al: Evidence for multiple structural genes for g chains of human fetal hemoglobin. Proc Natl Acad Sci USA 60:537, 1968.

Gelin BR, Lee AW-M, Karplus M: Hemoglobin structural tertiary change on ligand binding: Its role in the cooperative mechanism. J Mol Biol 171:489, 1983.

Arnone A: X-ray diffraction study of binding of 2,3-diphosphoglycerate to human deoxyhemoglobin. Nature 237:146, 1972.

Baldwin JM: The structure of human carbonmonoxyhaemoglobin at 2.7 Å resolution. J Mol Biol 136:103, 1980.

Baldwin J, Chothia C: Haemoglobin: The structural changes related to ligand binding and its allosteric mechanism. J Mol Biol 129:175, 1979.

Eisenberger P, Shulman RG, Kincaid BM, Brown GS, Ogawa S: Extended x-ray absorption fine structure determination of iron nitrogen distances in haemoglobin. Nature 274:30, 1978.

Warshel A: Energy-structure correlations in metalloporphyrins and the control of oxygen binding by hemoglobin. Proc Natl Acad Sci USA 74:1789, 1977.

Hsia CCW: Respiratory function of hemoglobin. N Engl J Med 338:239 1998.

Comroe JH Jr: Physiology of Respiration, p 161. Yearbook, Chicago, 1965.

Benesch RE, Benesch R: Mechanisms of interaction of red cell organic phosphates with hemoglobin. Adv Protein Chem 28:211, 1974.

Adair GS: The hemoglobin system. VI. The oxygen dissociation curve of hemoglobin. J Biol Chem 63:529, 1925.

Gibson QH: The reaction of oxygen with hemoglobin and the kinetic basis of the effect of salt on binding of oxygen. J Biol Chem 245:3285, 1970.

MacQuarrie R, Gibson QH: Use of a fluorescent analogue of 2,3-diphosphoglycerate as a probe of human hemoglobin conformation during carbon monoxide binding. J Biol Chem 246:5832, 1971.

Cassoly R, Gibson QH: Conformation, co-operativity and ligand binding in human hemoglobin. J Mol Biol 91:301, 1975.

Gibson QH: The photochemical formation of a quickly reacting form of haemoglobin. Biochem J 71:293, 1959.

Sharma VS, Geibel JF, Ranney HM: “Tension” on heme by the proximal base and ligand reactivity: Conclusions drawn from model compounds for the reaction of hemoglobin. Proc Natl Acad Sci USA 75:3747, 1978.

Moncada S, Palmer RM, Higgs EA: Nitric oxide: Physiology, pathophysiology and pharmacology. Pharmacol Rev 43:109, 1991.

Friebe A, Keosling D: Mechanism of YC-1 induced activation of soluble guanyl cyclase. Mol Pharmacol 53: 123–127, 1998.

Stamler JS, Jia L, Eu JP, et al: Blood flow regulation by S-nitrosohemoglobin in the physiological oxygen gradient. Science 276:2034, 1997.

Albitar M, Peschle C, Liebhaber SA: Theta, zeta, and epsilon globin messenger RNAs are expressed in adults. Blood 74:629, 1989.

Hsu S-L, Marks J, Shaw J-P, et al: Structure and expression of the human globin gene. Nature 331:94, 1988.

Brittain T, Hofmann OM, Watmough NJ, Greenwood C, Weber RE: A two-state analysis of co-operative oxygen binding in the three human embryonic haemoglobins. Biochem J 326:299, 1997.

Holmquist WR, Schroeder WA: A new N-terminal blocking group involving a Schiff base in hemoglobin A1c. Biochemistry 5:2489, 1966.

Rahbar S: An abnormal hemoglobin in red cells of diabetics. Clin Chim Acta 22:296, 1968.

Trivelli LA, Ranney HM, Lai H: Hemoglobin components in patients with diabetes mellitus. N Engl J Med 284:353, 1971.

Koenig RJ, Peterson CM, Jones RL, Saudek C, Lehrman M, Cerami A: Correlation of glucose regulation and hemoglobin A1c in diabetes mellitus. N Engl J Med 295:417, 1976.

Bunn HF, Haney DN, Kamin S, Gabbay KH, Gallop PM: The biosynthesis of human hemoglobin A1c: Slow glycosylation of hemoglobin in vivo. J Clin Invest 57:1652, 1976.

Koenig RJ, Blobstein SH, Cerami A: The structure of carbohydrate of hemoglobin A1c. J Biol Chem 252:2992, 1977.

Schroeder WA, Cua JT, Matsuda G, Fenninger WD: Hemoglobin F1, an acetyl-containing hemoglobin. Biochim Biophys Acta 63:532, 1962.

Fluckinger R, Harmon W, Meier W, et al: Hemoglobin carbamylation in uremia. N Engl J Med 304:823, 1981.
Copyright © 2001 McGraw-Hill
Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn
Williams Hematology


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