Structure and function of normal hemoglobins

INTRODUCTION — 

This topic discusses the structure and function of the normal human hemoglobins, the main component of red blood cells, which are responsible for oxygen delivery.

Separate topic reviews discuss fetal hemoglobin (Hb F) and abnormal hemoglobin variants associated with clinical disorders:

●Hb F – (See "Fetal hemoglobin (Hb F) in health and disease".)

●Sickle hemoglobin (Hb S) – (See "Pathophysiology of sickle cell disease".)

●Unstable hemoglobins – (See "Unstable hemoglobin variants".)

●Abnormal oxygen affinity hemoglobins – (See "Hemoglobin variants that alter hemoglobin-oxygen affinity".)

●Other variants – (See "Hemoglobin variants including Hb C, Hb D, and Hb E".)

GENERAL BACKGROUND

Fundamental insights in molecular biology — The study of hemoglobins, both normal and abnormal, has provided fundamental insights into gene structure, gene expression, structure-function relationships of proteins in general, and particularly the molecular basis of oxygen transport.

●Molecular basis of disease – The discovery that sickle hemoglobin (Hb S) has an abnormal electrophoretic mobility began the era of molecular medicine [1]. (See "Methods for hemoglobin analysis and hemoglobinopathy testing", section on 'Gel-based electrophoresis'.)

●Gene regulation – With the advent of recombinant DNA technology, research on hemoglobin provided early and important information about the organization and regulation of genes as well as insights as to how ontogeny affects gene expression [2]. (See 'Globin genes' below.)

●Gene-based therapies – Sickle cell disease and beta thalassemia are the first hereditary diseases to be cured by CRISPR/Cas9 gene editing and one of the first to be treated with gene therapy. (See "Overview of gene therapy, gene editing, and gene silencing" and "Curative therapies in sickle cell disease including hematopoietic stem cell transplantation and gene therapy", section on 'Gene therapies'.)

Proteins with hemoglobin-like function (hemoglobin motifs) exist in the most ancient unicellular plants and animals. They have evolved over hundreds of millions of years into gas transport proteins through the processes of gene duplication, conversion, divergence, and inactivating mutations [3].

Globin genes — Human globin genes are located in two independently regulated gene clusters on separate chromosomes (figure 1):

●Alpha-like genes – These are located near the telomere of the short arm chromosome 16. The two alpha globin genes (HBA2, HBA1, also referred to as the alpha globin loci 2 and 1) code for identical proteins but have some differences in the flanking regions (figure 1). The zeta gene (HBZ) is expressed mainly in the embryo and is the alpha-like component of embryonic hemoglobins.

●Beta-like genes – These are located near the tip of the short arm of chromosome 11. The single beta globin gene (HBB, also referred to as the hemoglobin beta locus) is present in a gene cluster that also contains an embryonic beta-like gene epsilon gene (HBE1), the duplicated and nearly identical fetal, or gamma globin genes (HBG2, HBG1), and the poorly expressed adult delta globin gene (HBD) (figure 1).

Two genes in the alpha-like gene cluster, the mu (HBM) and theta genes (HBQ1), do not partake in hemoglobin formation.

Globin gene expression is developmentally regulated, with different types of hemoglobins (consisting of different alpha-like and beta-like globins) predominating in the embryo, fetus, and adult [4]. (See 'Principles of hemoglobin switching and clinical significance' below.)

All human hemoglobin genes contain three exons separated by two introns. During messenger RNA (mRNA) processing, introns are spliced out, a 5' cap structure added, and the 3' end polyadenylated to make a mature contiguous globin mRNA that can be translated into globin. Exons might encode distinct functional domains of the molecule.

Types of globin gene variants — The types of variants determine the effect on hemoglobin and the clinical consequences:

●Thalassemias – Pathogenic variants that interfere with the splicing of introns or the accumulation of a mature mRNA can reduce production of one or the other globin chain, resulting in an imbalance of globin chains, which causes thalassemia. (See "Molecular genetics of the thalassemia syndromes", section on 'Variants affecting pre-mRNA splicing' and "Molecular genetics of the thalassemia syndromes", section on 'Altered mRNA translation and stability'.)

Alpha gene triplication can increase the severity of beta thalassemia in an individual who has a pathogenic variant on one beta globin allele [5,6].

●Variants that affect hemoglobin structure

•Mutations in exons can give rise to variant hemoglobins such as Hb S, Hb C, Hb D, or Hb E. (See "Pathophysiology of sickle cell disease", section on 'Genetics' and "Hemoglobin variants including Hb C, Hb D, and Hb E".)

•M hemoglobins alter the redox state of heme. (See 'Heme' below and "Methemoglobinemia", section on 'Hemoglobin M disease and cytochrome b5 deficiency'.)

•Some variants increase or decrease hemoglobin affinity for oxygen. (See "Hemoglobin variants that alter hemoglobin-oxygen affinity".)

•Other variants affect hemoglobin assembly or affinity for other molecules. (See "Unstable hemoglobin variants".)

●Variants that affect heme synthesis

•Mutations affecting heme biosynthetic enzymes can cause porphyria. (See "Porphyrias: Overview of classification and evaluation", section on 'Genes, enzymes, and intermediates'.)

•Variants affecting the first gene involved in heme synthesis (ALAS2) and the final gene (FECH) can cause sideroblastic anemia. (See "Causes and pathophysiology of the sideroblastic anemias", section on 'Heme synthesis'.)

Hemoglobin structure — Hemoglobin is a 64.4 kd heterotetramer consisting of two pairs of globin polypeptide chains. From birth onward, it consists of one pair of alpha chains and one pair of non-alpha chains, each chain surrounding a heme moiety. (See 'Heme' below.)

The globin composition is as follows:

●Fetal hemoglobin (Hb F) – Gamma2/alpha2

●Adult hemoglobin (Hb A) – Beta2/alpha2

●Hemoglobin A₂ – Delta2/alpha2

The tetrameric globular structure of hemoglobin is adapted to the physiology of complex organisms and their needs for regulation of oxygen delivery far better than the primitive globins like hemocyanin or erythrocruorin.

Heme

●Iron redox state – Heme (iron protoporphyrin IX) is a complex molecule in which a single ferrous (Fe⁺⁺) iron ion is surrounded by and coordinately bound to a protoporphyrin IX ring. If the iron is oxidized to the ferric state (Fe⁺⁺⁺), the protein is called methemoglobin, and the affected molecule does not bind oxygen. (See "Methemoglobinemia", section on 'What is methemoglobin?'.)

Some hemoglobin variants (Hb Ms) allow iron oxidation causing asymptomatic methemoglobinemia. (See "Methemoglobinemia", section on 'Hemoglobin M disease and cytochrome b5 deficiency'.)

●Sites of binding to globin – Heme iron is linked covalently to a histidine at the eighth residue of the F helix (His F8), at residue 87 of the alpha chain, and residue 92 of the beta chain (figure 2). Genetic variants that result in changes in some of the amino acids surrounding heme result in unstable hemoglobins. (See "Unstable hemoglobin variants", section on 'Altered heme-globin interactions'.)

Heme can bind oxygen but requires globin for the essential properties of cooperativity that allow oxygen binding in the lungs and delivery to the tissues. (See 'Oxygenation and deoxygenation' below.)

●Locations for heme synthesis – Heme synthesis occurs in all tissues but is especially prominent in bone marrow (used for synthesis of hemoglobin) and liver (used for hepatic cytochrome production). Synthesis occurs in the cytoplasm and mitochondria (figure 3). (See "Porphyrias: Overview of classification and evaluation", section on 'Normal heme biosynthesis'.)

●Sources of iron – Absorption of iron from the gastrointestinal tract and recycling of heme iron is discussed separately. (See "Regulation of iron balance", section on 'Systemic iron homeostasis'.)

Globin chains — The hemoglobin tetramer is a globular molecule (5.0 x 5.4 x 6.4 nm) with a single axis of symmetry [7]. The polypeptide chains are folded such that the four heme groups lie in clefts on the surface of the molecule equidistant from each other.

The six globin chains participating in hemoglobin formation are designated by Greek letters alpha through zeta. Hb A (alpha2/beta2) is composed of two alpha globin chains and two beta globin chains. (See 'Hemoglobin structure' above.)

●Amino acid length

•Alpha globin chains contain 141 amino acid residues.

•All beta-like chains contain 146 residues.

●Alpha helix – Approximately 75 percent of hemoglobin is in the form of an alpha helix. The nonhelical stretches permit folding of the polypeptide upon itself into the three-dimensional globin fold. Individual residues can be assigned to one of eight helices (A to H) or to adjacent nonhelical stretches.

●Secondary, tertiary, and quaternary folding – The primary amino acid structure of the constituent globin chains dictates the arrangement of the secondary helical and nonhelical sections and the tertiary globin fold. The quaternary structure of two pairs of unlike globin chains is the basis of the ability of hemoglobin to rapidly bind oxygen in the lungs and unload it in the tissues.

Single chain globins like muscle myoglobin (MB), cytoglobin (CYPG), and neuroglobin (NGB) are found in different tissues, bind oxygen with high affinity, and lack cooperativity. (See 'Cooperativity' below.)

Regulation of hemoglobin assembly — Newly synthesized alpha globin is unstable and requires a chaperone (alpha hemoglobin stabilizing protein [AHSP]) for its protection before it forms alpha-non-alpha dimers. Because of this instability, an alpha globin chain tetramer (alpha4) does not appear to occur [8].

However, when alpha chains are reduced or absent, as occurs in alpha thalassemia, beta globin and gamma globin can homotetramerize, forming Hb H (beta4) and hemoglobin Barts (gamma4). These homotetramers lack cooperativity and are poor oxygen transporters. (See "Pathophysiology of thalassemia", section on 'Alpha thalassemia'.)

●Stable alpha/beta heterodimers – When both alpha and beta globin are present, electrostatic interactions lead to formation of highly stable alpha/beta dimers. These self-associate into much less stable hemoglobin tetramers that can readily dissociate.

●Mixed hybrid tetramers with Hb F – When other globin chains are present, such as gamma globin, mixed hybrid tetramers can form, such as alpha2/betaS-gamma, in mixtures of Hb F and Hb S. This accounts in large part for the anti-sickling properties of Hb F [9,10]. (See "Fetal hemoglobin (Hb F) in health and disease", section on 'Impact of Hb F on sickle cell disease severity'.)

If the Hb F concentration in a sickle red blood cell is 30 percent, approximately 50 percent of tetramers are Hb S, 10 percent are Hb F, and 40 percent are mixed hybrid alpha2/betaS-gamma tetramers. Separation of hemoglobin fractions that are used clinically like high performance liquid chromatography (HPLC) or capillary electrophoresis cannot detect these or other mixed hybrid tetramers [11].

●Solubility – Residues that have charged side groups, such as lysine, arginine, and glutamic acid, lie on the surface of the molecule in contact with the surrounding water solvent. Exposure of the hydrophilic (charged) amino acids to the aqueous milieu is an important determinant of the solubility of hemoglobin within the red blood cell and helps prevent hemoglobin from precipitating. (See "Unstable hemoglobin variants", section on 'Genetic variants and their effect on protein structure'.)

HEMOGLOBIN FUNCTION — 

The principal physiologic function of hemoglobin is to bind, carry, and deliver oxygen from the lungs to the tissues and to return carbon dioxide from tissues to the lungs. (See 'Oxygenation and deoxygenation' below.)

Another function, delivery of nitric oxide and regulation of vasomotor tone, has been postulated. (See 'Nitric oxide transport' below.)

Although many other functions of hemoglobin have been described, none are pertinent to its respiratory function [12-14].

Oxygenation and deoxygenation — Oxygenation and deoxygenation of hemoglobin occurs at the heme iron. Upon deoxygenation, the hemoglobin molecule undergoes a marked change in conformation, as the beta chains rotate apart by approximately 0.7 nm.

●T versus R configuration – Deoxyhemoglobin is stabilized in a constrained or tense (T) configuration by inter-subunit and intra-subunit salt bonds. These salt bonds are responsible for the Bohr effect and for the binding of 2,3-BPG, both of which modify oxygen affinity. Crystallographic studies suggest that the switch between the T and R states is more complex than described by the two-state model [15]. (See 'Oxygen affinity' below and "Hemoglobin variants that alter hemoglobin-oxygen affinity", section on 'Pathophysiology'.)

Upon the addition of oxygen, these salt bonds are sequentially broken, and the fully oxygenated hemoglobin is in the relaxed (R) configuration. In this state, there is less bonding energy between the subunits, and the oxygenated molecule can dissociate reversibly, forming two alpha beta dimers. When cell-free hemoglobin is present in the circulation, these dimers bind to haptoglobin and can be filtered at the glomerulus. (See "Diagnosis of hemolytic anemia in adults", section on 'High LDH and bilirubin; low haptoglobin'.)

●Hemoglobin-oxygen dissociation curve – Oxygenation of hemoglobin as a function of the oxygen concentration is expressed by a sigmoid hemoglobin-oxygen association/dissociation curve, which has two characteristics (figure 4):

•The shape of this curve is due to a phenomenon known as cooperativity, whereby partial saturation of hemoglobin with oxygen increases the affinity of the remaining hemes for oxygen. The homotetrameric hemoglobins H and hemoglobin Barts lack the cooperativity of normal heterotetrameric hemoglobins and have a hyperbolic hemoglobin-oxygen dissociation curve. (See 'Cooperativity' below.)

•The affinity of hemoglobin for oxygen can be modified (ie, shifted to the right or left) by various exogenous factors such as pH, 2,3-BPG, and temperature, as discussed below. (See 'Oxygen affinity' below.)

Cooperativity — The phenomenon of cooperativity is due to the T (tense) form of hemoglobin having a lower affinity for ligands such as oxygen and carbon monoxide (CO) than the R (relaxed) form. Heme-heme interaction, a function of the tetrameric nature of hemoglobin, implies that the four heme groups do not undergo simultaneous oxygenation or deoxygenation; instead, the oxygenation state of each individual heme molecule influences the binding of oxygen to the other heme groups.

The totally deoxygenated hemoglobin tetramer is slow to bind the first oxygen molecule, but the reaction with oxygen accelerates as oxygenation proceeds. At some point during the sequential addition of oxygen to the four hemes of the molecule, a transition from the T to the R conformation occurs. At this point, the oxygen affinity of the partially liganded molecule increases markedly [16]. When two or three molecules of oxygen are bound, the alpha1/beta2 interface is sufficiently destabilized to flip the quaternary structure from T to R, thereby increasing the affinity of the remaining hemes for oxygen. (See 'Oxygenation and deoxygenation' above.)

Oxygen affinity — Oxygen affinity is usually designated by the P₅₀, which is the partial pressure of oxygen at which hemoglobin is 50 percent saturated, as illustrated in the classic hemoglobin-oxygen dissociation curve (figure 4). The P₅₀ is normally 27 mmHg (27 torr) at 37°C and pH 7.4.

Oxygen affinity is modulated by pH, CO₂, 2,3-BPG, and temperature, as discussed below. These changes are important in the body's ability to adapt to hypoxia and anemia. (See 'pH' below and 'Carbamino adducts' below and '2,3-bisphosphoglycerate' below and 'Temperature' below.)

●Right shift – A shift to the right in the hemoglobin-oxygen association/dissociation curve, with a higher P₅₀, indicates reduced oxygen affinity, increased oxygen delivery to the tissues, and requirement for a higher partial pressure of oxygen to saturate hemoglobin in the lungs.

Conditions that shift the curve rightward (reduced oxygen affinity, increased delivery to tissues) include low pH, high temperature, and high 2,3-BPG (figure 4). These conditions occur in states of increased oxygen demand such as fever and increased metabolism.

Some variant hemoglobins have right-shifted curves. Increased oxygen release causes anemia and cyanosis. (See "Hemoglobin variants that alter hemoglobin-oxygen affinity".)

●Left shift – Conversely, a shift to the left, with a lower P₅₀, indicates increased oxygen affinity, decreased oxygen delivery to tissues, and requirement for a lower partial pressure of oxygen to saturate hemoglobin.

Conditions that shift the curve leftward (increased oxygen affinity, reduced oxygen delivery to tissues) include high pH, low temperature, low 2,3-BPG, and high Hb F (figure 4).

Some variant hemoglobins have left-shifted curves. Decreased oxygen release stimulates erythropoietin production and causes erythrocytosis. (See "Hemoglobin variants that alter hemoglobin-oxygen affinity".)

pH — The oxygen affinity of hemoglobin increases as a function of pH over a range of 6.0 to 8.5, a phenomenon known as the Bohr effect [17]. A corollary of this relationship is that deoxyhemoglobin binds protons more strongly than does oxyhemoglobin. Under physiologic conditions, a molecule of hemoglobin releases approximately 2.8 protons upon oxygenation.

A substantial portion of the Bohr effect is due to an intra-subunit salt bond between the positively charged imidazole of beta146 histidine and the negatively charged carboxyl of beta94 aspartate [18,19]. This salt bridge helps to stabilize the deoxy conformation. When hemoglobin is oxygenated, these bonds are broken, and protons are released.

There are two major physiologic consequences of the Bohr effect. First, oxygen is more easily released at the tissue level due to a higher carbon dioxide (CO₂) tension and a lower pH. Second, oxygen is more easily taken up in the pulmonary circulation where the pH is higher due to the efflux of CO₂.

Carbamino adducts — CO₂ can react with free amino groups at the N termini of the alpha and beta chains to form carbamino complexes. Deoxyhemoglobin forms these complexes more readily than oxyhemoglobin; thus, at any given pH, CO₂ lowers oxygen affinity.

Normally, approximately 10 percent of CO₂ is transported to the lungs in the form of carbamino hemoglobin [20].

2,3-bisphosphoglycerate — 2,3 BPG is a potent modulator of the affinity of hemoglobin for oxygen. It is synthesized from 1,3-BPG in the glycolytic pathway under the influence of the enzyme bisphosphoglycerate mutase (BPGM) (figure 5).

2,3 BPG is normally present in red blood cells at a concentration of approximately 5 mmol/L. It is a polyanion that binds strongly to deoxyhemoglobin in a 1:1 molar ratio but weakly to oxyhemoglobin, as described by the following reaction:

Binding takes place in the central cavity of the hemoglobin molecule, between the two beta chains, where the negative charges of 2,3-BPG are neutralized by the beta amino-terminal histidine, beta82 lysine, and beta143 histidine. Binding to deoxyhemoglobin helps to stabilize the tense (T) structure of hemoglobin and decrease oxygen affinity. In oxyhemoglobin, the H helices of the beta chains are insufficiently spread apart to permit firm binding of 2,3-BPG. (See "Hemoglobin variants that alter hemoglobin-oxygen affinity", section on 'Pathophysiology'.)

●Increased 2,3-BPG – Increased 2,3-BPG shifts the hemoglobin oxygen dissociation curve to the right, decreases oxygen affinity, and increases the delivery of oxygen to tissues (figure 4).

●Decreased 2,3-BPG – Decreased 2,3-BPG shifts the hemoglobin oxygen dissociation curve to the left, increases oxygen affinity, and decreases the delivery of oxygen to tissues.

•Genetic causes – Pathogenic variants in the BPGM gene that decrease 2,3-BPG production and reduce tissue oxygen delivery can result in a compensatory increase in erythropoietin, stimulating erythropoiesis and producing autosomal dominant erythrocytosis [21-23]. (See "Molecular pathogenesis of congenital erythrocytoses and polycythemia vera", section on '2,3-bisphosphoglycerate deficiency'.)

•RBC storage – During storage of RBC units, 2,3-BPG levels decline, increasing oxygen affinity. Transfusion of stored blood is accompanied by restoration of 2,3 BPG within 12 to 24 hours [24]. Prolonged storage also resulted in irreversible changes affecting the secondary and quaternary structures of hemoglobin, with subsequent irreversible locking of the molecule in the R state. This appears to be due to formation of beta sheets and a decrease in alpha helices stabilized by strong intermolecular hydrogen bonding. Despite these findings, longer storage duration has not been shown to affect transfusion outcomes. (See "Practical aspects of red blood cell transfusion in adults: Storage, processing, modifications, and infusion", section on 'Changes during in vitro storage'.)

•Pyruvate kinase activators – Small molecule agonists of the enzyme pyruvate kinase (PK) increase enzyme activity in some patients with PK deficiency [25]. By increasing PK activity, these agonists also reduce the levels of 2,3 BPG. The resulting increased oxygen affinity reduces the polymerization of sickle hemoglobin and is being studied as a treatment for sickle cell disease (SCD). (See "Pyruvate kinase deficiency", section on 'Mitapivat for symptomatic anemia' and "Investigational pharmacologic therapies for sickle cell disease", section on 'Pyruvate kinase activation (mitapivat, etavopivat)'.)

PK activators are also being studied in beta thalassemia, where one study found where hemoglobin is increased by approximately 1 g/dL in one-half of the treated patients [26]. In thalassemia, an increase in ATP production may be a more important mechanism of action of the PK activators [27]. (See "Management of thalassemia", section on 'Mitapivat'.)

Temperature — Oxygen affinity varies inversely with temperature. The decrease in oxygen affinity at elevated body temperature helps to unload oxygen at the tissue at a time when oxygen requirement is likely to be increased.

The position and shape of the oxyhemoglobin dissociation curve define the amount of oxygen that is unloaded at the tissue at any given blood flow and hemoglobin concentration. When the oxyhemoglobin dissociation curve is right-shifted, red blood cells have enhanced oxygen release when going from a normal arterial pO₂ (95 mmHg) to a normal mixed venous pO₂ (40 mmHg). This is because with lower pO₂, a steeper portion of the curve is encompassed.

Nitric oxide transport — Nitric oxide (NO) is a potent vasodilator. The oxidized nitrosyl derivative of NO can form adducts with proteins, including sulfhydryl groups on cysteine residues. Hemoglobin contains a highly conserved cysteine (beta93cys) on the beta subunit, close to the heme pocket. The sulfhydryl group of this conserved cysteine has free access to modification when hemoglobin is oxygenated but is sterically blocked when the molecule is deoxygenated [28].

It has been suggested that NO produced in the pulmonary circulation binds to the cysteine sulfhydryl group, forming the S-nitrosothiol (SNO) derivative. As blood traverses tissues and unloads oxygen, NO is ejected from hemoglobin and diffuses out of the red blood cell into the lumen of arterioles and capillaries, where it serves as a local vasodilating agent [29-34]. This effect may also serve to control the respiratory drive (figure 6). (See "Control of ventilation", section on 'Peripheral chemoreceptors'.)

There is controversy about whether SNO hemoglobin is part of a three-gas respiratory cycle of O₂/NO/CO_2, and a critical mediator of tissue blood flow and oxygen delivery, with evidence pro and con [34,35]. It will be difficult to critically assess whether there is oxygen-linked net release of NO from red blood cells during the rapid transit time from arteries through the precapillary circulation (less than one-half of a second).

DIFFERENT TYPES OF HEMOGLOBIN AT DIFFERENT STAGES OF LIFE

Principles of hemoglobin switching and clinical significance — The major globin genes are differentially expressed during embryonic and fetal development and after birth. This allows oxygen transport to flow in the direction needed to support embryonic development, followed by transfer from the placenta to the fetal circulation, followed by delivery of inhaled oxygen to the tissues after birth.

●Globin chains according to stage of life

•Embryo – The alpha-like chains are zeta and alpha. The beta-like chains are epsilon and gamma. (See 'Embryonic hemoglobins' below.)

•Fetus – Hb F is composed of alpha and gamma chains. (See 'Hb F' below and "Fetal hemoglobin (Hb F) in health and disease", section on 'Biology of Hb F'.)

•After birth – Approximately six months after birth and continuing into adulthood, the main hemoglobin is Hb A (alpha2/beta2), with a small percentage of Hb A₂ (alpha2/delta2) and Hb F. (See 'Adult hemoglobins (predominant from month 6 onward)' below.)

●Switching of alpha-like genes

During the transition from embryonic to fetal hemoglobin, the zeta globin gene is repressed as alpha globin gene expression begins. This switch is mediated by the major alpha globin gene enhancer (previously termed HS-40) (figure 1). The enhancer covers a region approximately 8 to 31 kilobases upstream of these genes and consists of five separate elements: R1, R2, R3, Rm, and R4. These are bound by many different transcription factors and interact with the alpha globin gene promoters [36].

From fetal development onward, there is no substitute for alpha globin in any of the normal hemoglobins (Hb F, Hb A, and Hb A₂). Thus, absence of alpha globin, as seen when all four alpha globin genes are inactive or deleted, producing alpha thalassemia hydrops fetalis, is incompatible with prolonged extrauterine life unless extraordinary measures such as regular transfusions or hematopoietic stem cell transplantation are taken. (See "Alpha thalassemia major: Prenatal and postnatal management", section on 'Management (prenatal and neonatal)'.)

●Switching of beta-like genes – The hemoglobin switch from HBE1 (encoding epsilon chains in embryonic hemoglobins) to HBG2 and HBG1 (encoding gamma chains in Hb F) to HBB (encoding beta chains in Hb A) is discussed in detail separately. (See "Fetal hemoglobin (Hb F) in health and disease", section on 'Hemoglobin switching and downregulation of Hb F expression'.)

Reversing this switch is a therapeutic goal for treating sickle cell disease and beta thalassemia. (See "Curative therapies in sickle cell disease including hematopoietic stem cell transplantation and gene therapy", section on 'Gamma globin upregulation (including exa-cel, Casgevy)' and "Hematopoietic stem cell transplantation and other curative therapies for transfusion-dependent thalassemia".)

Impact on age of presentation of hemoglobinopathies — Because different globin chains are expressed at different developmental stages, disorders affecting a particular globin gene (alpha-like or beta-like) present at different ages:

●Alpha globin disorders (presentation in utero) – Variants in the alpha globin genes (HBA2, HBA1) are phenotypically apparent early in development (ie, after approximately the 14^th week of gestation). Any associated phenotype is likely to persist throughout fetal, neonatal, and adult life.

●Gamma globin disorders (only affect the fetus and neonate) – Variants in the gamma globin genes (HBG2, HBG1) appear in the fetus, can linger into the neonatal period, and, if severe, can cause fetal loss. After the neonatal period, gamma globin gene variants are unlikely to be associated with a phenotype, as Hb F levels fall to less than 1 percent of normal during the first year of life.

●Delta globin variation (clinically silent) – Delta globin gene (HBD) variants become detectable late in the first year of life, as this gene is minimally expressed during intrauterine development. Such variants have no ill effects as the delta gene is expressed at very low levels.

●Beta globin disorders (presentation several months after birth) – Variants in the beta globin gene (HBB), such as those encoding Hb S or beta thalassemia, become apparent only after the switch from Hb F (alpha2/gamma2) to Hb A (alpha2/beta2). They are compatible with fetal survival and may not become apparent until the middle to late months of the first year of life. (See "Overview of the clinical manifestations of sickle cell disease".)

Embryonic hemoglobins

Globin chain composition and timing of expression (embryonic Hbs) — In early development, between weeks 4 and 14 of gestation, the human embryo synthesizes three distinct hemoglobins. They are produced by large, primitive, nucleated erythroblasts in the yolk sac.

These embryonic hemoglobins are, in their order of appearance [37]:

●Hb Gower I – Zeta2/epsilon2

●Hb Portland – Zeta2/gamma2

●Hb Gower II – Alpha2/epsilon2

Embryonic hemoglobins that contain zeta globin chains (encoded by the HBZ gene) or epsilon globin chains (encoded by the HBE1 gene) are primarily the product of yolk sac erythroblasts and are detectable only during the very earliest stages of embryogenesis, except for trace amounts in severe alpha thalassemia. By approximately the 14^th week of gestation, after establishment of erythropoiesis in the fetal liver and spleen, these embryonic hemoglobins are completely replaced by Hb F.

Alpha globin variants that also delete these embryonic alpha-like zeta chains have an earlier presentation of alpha thalassemia than variants that do not affect zeta chains. (See "Alpha thalassemia major: Prenatal and postnatal management", section on 'Timing of onset'.)

Oxygen carrying properties (embryonic Hbs) — Embryonic hemoglobins have evolved to permit oxygen transport from amniotic fluid (a low oxygen environment) to the cells of the developing embryo, prior to establishment of a placental circulation. They are characterized by increased oxygen binding (P₅₀ 4 to 12 mmHg) and slightly reduced cooperativity (h = 1.9 to 2.3) (figure 4). This serves to provide needed oxygen during embryogenesis [38]. These features of embryonic hemoglobins might be a consequence of the weak subunit assembly into tetrameric hemoglobin relative to Hb A or Hb F [39].

●Hb Gower I and II – Organic phosphates lower the oxygen affinities of Hb Gower 1 and Gower II. This demonstrates that the zeta and epsilon chains do not completely interfere with the binding of 2,3 BPG.

According to crystallographic studies, the tertiary structure of the alpha chain in Hb Gower II is identical to Hb A and Hb F [40].

The epsilon chain has a structure very similar to the beta chain (lysine rather than threonine at epsilon87), with small differences in the N terminus and the alpha helix. The Cl- binding sites involve the polar residues within the central cavities; Hb Gower II has reduced sensitivity to chloride irons (Cl-). Mutagenesis identified the beta77 replacement (histidine—>asparagine) in the epsilon chain as the origin of its lower sensitivity towards chloride ions [41]. This allows oxygen exchange from the mother to the late embryo under physiological conditions.

This difference in the epsilon chain is reminiscent of the gamma and delta chains, which also have substitutions at this site and which are both anti-sickling hemoglobins.

●Hb Portland – The amino acid threonine at position 38 in Hb Portland is universally conserved in all mammals; mutagenesis of this amino acid to glutamine changes the equilibrium properties of the T state [42]. Hb Portland does not bind phosphates effectively since its central cavity is formed by gamma chains.

Hb F — Details of the production and biology of Hb F (fetal hemoglobin), along with supporting references, are presented separately. (See "Fetal hemoglobin (Hb F) in health and disease".)

Summarized briefly, the key points include:

●Composition and properties – Hb F consists of two alpha chains and two gamma chains (alpha2/gamma2).

•There are two genes for gamma globin chains, resulting from gene duplication (figure 1). There is only a single amino acid difference between the globin chains they produce, and they are functionally similar [43].

•Hb F in fetal red blood cells has a considerably higher oxygen affinity than Hb A in adult red blood cells (figure 4); this is because Hb F binds poorly to 2,3-BPG [44,45]. This difference facilitates oxygen transfer from maternal Hb A in the placenta to Hb F in the fetal circulation, providing adequate oxygen for fetal development.

•Hb F is a much more stable hemoglobin tetramer than Hb A [46]. It dissociates into dimers less readily than Hb A, is resistant to losing hemes, and is remarkably resistant to denaturation at extremes of pH.

•Gamma globin genes are distinct from the beta globin gene and lack the gene variants (point mutations, deletions) that cause sickle cell disease and beta thalassemia.

-The main mechanism of hydroxyurea is thought to be via increasing Hb F production. (See "Hydroxyurea use in sickle cell disease", section on 'Mechanism of action'.)

-Gene therapy and gene editing approaches to increasing Hb F levels are in various stages of study or approval for treating these conditions. (See "Curative therapies in sickle cell disease including hematopoietic stem cell transplantation and gene therapy", section on 'Gamma globin upregulation (including exa-cel, Casgevy)' and "Hematopoietic stem cell transplantation and other curative therapies for transfusion-dependent thalassemia", section on 'Gene therapy and gene editing'.)

●Timing of expression

•Hb F becomes the predominant hemoglobin of the fetus from the eighth week of gestation onward.

•By 28 weeks of gestation, Hb F constitutes approximately 90 percent of total hemoglobin; by 38 weeks, it decreases to approximately 60 percent. Red blood cells in newborns contain approximately 60 percent Hb F and 40 percent Hb A.

•Several months following birth, Hb F becomes a minor hemoglobin, constituting <1 percent of total hemoglobin. The distribution among red blood cells is uneven, with only 0.1 to 7 percent of red blood cells containing detectable levels of Hb F [47,48].

●Regulation

•Transcriptional repressors regulate the transition from Hb F to Hb A. (See "Fetal hemoglobin (Hb F) in health and disease", section on 'Hemoglobin switching and downregulation of Hb F expression'.)

•Three main quantitative trait loci (QTL) control the levels of Hb F expression: HBS1L-MYB, BCL11A, and the HBG2 Xmn1 polymorphism in the HBG2 promotor. (See "Fetal hemoglobin (Hb F) in health and disease", section on 'Quantitative trait loci associated with HBG expression'.)

●Quantification – Hb F is easily separated from Hb A by a number of protein separation techniques including gel electrophoresis (figure 7), isoelectric focusing (figure 8), HPLC (figure 9), or capillary electrophoresis (figure 10).

Hb F is increased in some hemoglobinopathies including beta thalassemia and sickle cell disease. (See "Fetal hemoglobin (Hb F) in health and disease", section on 'Conditions causing increased Hb F'.)

●Hereditary persistence of fetal hemoglobin (HPFH) – HPFH is an asymptomatic condition in which high levels of Hb F persist into adulthood (5 to 40 percent, depending on the underlying genetic variants). Details are presented separately. (See "Fetal hemoglobin (Hb F) in health and disease", section on 'Hereditary persistence of fetal hemoglobin (HPFH)'.)

Non-genetic conditions can also cause transient elevations in Hb F; these are discussed separately. (See "Fetal hemoglobin (Hb F) in health and disease", section on 'Secondary increases in Hb F'.)

Adult hemoglobins (predominant from month 6 onward)

Hb A — Hb A (alpha2/beta2) is the major adult hemoglobin, constituting approximately 40 percent of total hemoglobin at birth and increasing to 95 to 98 percent by older childhood.

Hb A exhibits cooperative binding to oxygen, which can be modified by pH, 2,3-BPG, and temperature. (See 'Cooperativity' above and 'Oxygen affinity' above.)

Methods for separating Hb A from other hemoglobins are discussed separately. (See "Methods for hemoglobin analysis and hemoglobinopathy testing", section on 'Protein chemistry methods' and "Methods for hemoglobin analysis and hemoglobinopathy testing", section on 'Point-of-care assays'.)

Causes of low Hb A (anemia) are discussed separately. (See "Approach to the child with anemia" and "Diagnostic approach to anemia in adults".)

Hb A2 — Approximately 2 to 3 percent of the hemoglobin in adult red blood cells is hemoglobin A₂ (Hb A₂; alpha2/delta2) [49-52].

●Composition and properties

•Delta globin chains are identical in sequence to beta globin chains in all but 10 of 146 residues, and Hb A₂ functional behavior is very similar to that of Hb A [20]. Minor differences in the functional properties of HbA₂ have no clinical significance.

•Hb A₂ is more positively charged than Hb A [53]. The positively charged delta globin chains have reduced affinity (relative to beta globin chains) for the positively charged alpha globin chains [10]. This in turn reduces the amount of Hb A₂, which forms more slowly than Hb A [54,55].

•Hb A₂ has slightly higher oxygen affinity than Hb A, while the Bohr effect, cooperativity, and response to 2,3 BPG are identical [56].

•Thermal stability of Hb A₂ is greater than that of Hb A, which may be due to the delta116 arg that can form a salt bridge with alpha114 pro, further increasing the stability of the alpha1/beta1 packing contact.

•Relative to Hb A, Hb A₂ has slightly increased susceptibility to autoxidation to methemoglobin, its hemichrome has increased stability, and its membrane binding is increased. The effects of very high levels of Hb A₂ could be deleterious [50].

•Hb A₂ has anti-sickling properties, similar to Hb F, and it contains the same position 87 threonine-glutamine substitution as Hb F [57]. Overexpression of Hb A₂, in mice carrying the sickle cell variant resulted in increased hemoglobin, reduced organ damage, and prolonged lifespan [58].

Gene therapy that takes advantage of the unaffected delta globin gene in individuals with sickle cell disease is under investigation. It is possible that, due to its positive charge, high levels of Hb A₂ might damage the red blood cell membrane [50]. (See "Curative therapies in sickle cell disease including hematopoietic stem cell transplantation and gene therapy", section on 'Delta globin gene'.)

•Hb A₂ is present in humans, apes, and New World monkeys. It is expressed in non-human primates at low levels, except in the Galago, which has an exceptionally altered delta gene sequence. Old World monkeys have a delta globin gene that has accumulated inactivating mutations and has become a pseudogene [59].

●Quantification

•Hb A₂ can be readily separated from Hb A by capillary electrophoresis (figure 10) or HPLC (figure 9).

●Regulation – Reduced synthesis of delta globin chains relative to beta globin chains might be the result of one or both of the following:

•The half-life of delta globin mRNA is less than one-third that of beta-globin mRNA, probably due to changes in the delta mRNA sequence in the 3' untranslated region. However, this mechanism alone does not account for the vastly reduced concentration of Hb A₂ compared with Hb A. Hb A and Hb A₂ have almost identical molecular stability, making it unlikely that differential posttranslational survival of the two molecules accounts for this difference [49].

•Genetic variation in the delta globin promotor (in the Kruppel-like factor 1 [Klf1] binding site [CACCC box]) might account for reduced synthesis of Hb A₂ [60]. The human delta globin gene can be activated in vivo by the insertion of a Klf1 binding site into the promoter [61]. Two studies have noted Hb A₂ levels in the borderline high range in individuals with a pathogenic variant in the KLF1 gene (3.5±0.2 percent, range: 3.1 to 4.0 in one study and 3.3±0.4 percent, range: 2.5 to 4.9 in the other) [51,62].

•A genome-wide association study implicated the HBS1L-MYB locus on chromosome 6q and a second locus on chromosome 11p surrounding HBB in controlling delta globin expression [63]. Other studies have identified an association with BCL11A on chromosome 2p and polymorphisms downstream of the beta globin gene cluster on chromosome 11; the latter effect was independent of Hb F [64].

●Causes of increased or decreased Hb A₂ – The table summarizes the main causes (table 1). These include some heritable and some acquired conditions [49-52].

•Increased Hb A₂ can be seen in beta thalassemia trait (5.4±0.4 percent, range: 4.5 to 6.2), a finding that is a useful diagnostic aid. When beta thalassemia is caused by deletions of the 5' portion of HBB, Hb A₂ levels can be as high as 7 to 9 percent. Hb A₂ is also slightly increased in megaloblastic anemia.

•Mildly increased Hb A₂ has been seen with some mild beta⁺ thalassemia variants and other globin variants.

•Decreased Hb A₂ can be seen in:

-Iron deficiency

-Alpha thalassemia

-Sideroblastic anemia

The decrease in Hb A₂ with iron deficiency explains why individuals with concomitant beta thalassemia and iron deficiency may have falsely normal (or lower than expected) levels of Hb A₂ and provides the rationale for obtaining iron studies prior to protein-based beta thalassemia testing [65,66]. (See "Diagnosis of thalassemia (adults and children)", section on 'Rule out iron deficiency'.)

POSTTRANSLATIONAL MODIFICATIONS

Glycation/glycosylation — When hemoglobin is analyzed by column chromatography or HPLC (high performance liquid chromatography), several minor components can be detected that have lower isoelectric points than Hb A (figure 9) [67,68].

These are designated A1a through A1e.

●Hb A1c – Hemoglobin A1c generally correlates with mean blood glucose values in individuals with normal red blood cell turnover (ie, without hemolysis). (See 'Role of Hb A1c in assessing blood glucose levels' below.)

Hb A1c accounts for approximately 3 percent of the hemoglobin in red blood cells of individuals with blood glucose in the normal range [69]. (See "Laboratory test reference ranges in adults", section on 'Hemoglobin, glycated (Hb A1c)'.)

Hb A1c differs from Hb A only at the N-terminal amino group of each beta chain, in which glucose is attached nonenzymatically by a keto-amine linkage [70,71].

●Hb A1b – Hb A1b is an adduct of pyruvate with the beta-N terminus [72].

●Approximately 5 percent of hemoglobin molecules have glucose linked to certain lysine residues. These adducts cannot be separated from unmodified hemoglobins by ordinary chromatography or electrophoresis, but they can be isolated by means of an affinity resin containing phenylboronate, which binds to sugar hydroxyl groups.

Over a period of weeks to months, glycated proteins can undergo further rearrangement reactions to form fluorescent advanced glycation end products [73].

Role of Hb A1c in assessing blood glucose levels — Since glucose becomes irreversibly attached to hemoglobin during the life of the red blood cell at a rate dependent upon the prevailing blood glucose, measurement of A1c has proven to be useful for screening for diabetes and for independent assessment of the degree of diabetic control. Hb A1c reflects the mean blood glucose over the previous six to eight weeks [74].

Individuals with untreated diabetes mellitus may have levels of hemoglobin A1c that are two to three times higher than the reference range [75].

●Hb A1c in diabetes screening – (See "Screening for type 2 diabetes mellitus and prediabetes", section on 'Glycated hemoglobin (A1C)'.)

●Hb A1c in diabetes monitoring – (See "Measurements of chronic glycemia in diabetes mellitus", section on 'Glycated hemoglobin (A1C)'.)

Limitations of Hb A1c in evaluating blood glucose values include:

●Hemolysis – The addition of sugar phosphates and other red blood cell metabolites to hemoglobin occurs slowly and continuously throughout the 120-day life span of the red blood cell. Consequently, hemolysis, which leads to more rapid turnover of red blood cells, will decrease levels of these modifications. As a result, the Hb A1c may underestimate mean blood glucose values.

●Variant hemoglobins – Depending upon the methodology used (eg, HPLC, immunoassay), erroneous values may occur in patients with high levels of Hb F or with abnormal hemoglobins such as Hb S or Hb Wayne [76].

●Other substances – Additional substances that interfere with Hb A1c test results are summarized on the website of the National Glycohemoglobin Standardization Program (NGSP) [77].

Details are presented separately. (See "Measurements of chronic glycemia in diabetes mellitus", section on 'Glycated hemoglobin (A1C)'.)

Other posttranslational modifications — Although glucose adducts are by far the most common and abundant type of chemical modification of hemoglobin, other small molecules are also capable of forming covalent linkages that may reflect significant metabolic perturbations. Examples include [78-80]:

●Cyanate adducts in patients with uremia

●Acetaldehyde adducts in individuals with excess alcohol intake

●Porphyrin-substituted hemoglobin

SUMMARY

●Structure – Hemoglobin studies have provided fundamental insights into gene structure, expression, and function. Human hemoglobins are heterotetramers containing two alpha-like chains and two beta-like chains, encoded by two independently-regulated gene clusters (figure 1). Each globin chain binds one heme molecule consisting of a protoporphyrin IX ring surrounding a ferrous iron. (See 'General background' above.)

●Function – Hemoglobin binds oxygen in the lungs and releases it to the tissues. This is possible because of cooperativity. Deoxygenated hemoglobin tetramers in a hemoglobin molecule bind oxygen slowly, but as oxygenation proceeds, oxygen binding to the other globins accelerates. This is illustrated in the sigmoidal shape of the hemoglobin oxygen dissociation curve (figure 4). (See 'Oxygenation and deoxygenation' above and 'Cooperativity' above and 'Oxygen affinity' above.)

•The curve shifts rightward (reduced oxygen affinity, increased delivery to tissues) with increased temperature and lower pH.

•The curve shifts leftward (increased oxygen affinity, reduced delivery to tissues) with lower temperature, higher pH, low 2,3-BPG (bisphosphoglycerate), and high fetal hemoglobin (Hb F).

●Changes across the lifespan – Globin genes are differentially expressed during embryonic and fetal development and after birth. Their different properties and oxygen affinities facilitate oxygen transfer through the amniotic fluid to the embryo, from the placenta to the fetus, and from the lungs to the tissues. The age at which different globin gene variants present clinically depends on the gene expression pattern of the affected gene. (See 'Principles of hemoglobin switching and clinical significance' above and 'Impact on age of presentation of hemoglobinopathies' above.)

•Embryonic hemoglobins – These are produced by yolk sac erythroblasts and include Hb Gower I (zeta2/epsilon2), Portland (zeta2/gamma2) and Gower II (alpha2/epsilon2). They are replaced by Hb F at approximately week 14 of gestation. (See 'Embryonic hemoglobins' above.)

•Fetal hemoglobin – Production of Hb F begins with establishment of erythropoiesis in the fetal liver and spleen, reaches approximately 90 percent by 28 weeks of gestation, and decreases to approximately 60 percent at term and to <1 percent within a few months after birth. Causes of high Hb F and strategies to increase Hb F therapeutically in sickle cell disease and thalassemia are discussed separately. (See 'Hb F' above and "Fetal hemoglobin (Hb F) in health and disease".)

•Adult hemoglobins – Hb A (alpha2/beta2) accounts for 95 to 98 percent of hemoglobin from approximately six months onward. Hb A₂ (alpha2/delta2) accounts for 2 to 3 percent, with increases or decreases in certain hereditary and acquired conditions (eg, decreased in iron deficiency; increased in beta thalassemia) (table 1). Increasing Hb A₂ is under investigation for beta hemoglobinopathies. (See 'Hb A' above and 'Hb A2' above.)

●Hemoglobin A1c – Hemoglobin A1c is Hb A with added glucose molecules. It accounts for approximately 3 percent of total hemoglobin in adult red cells and is increased in diabetes mellitus, especially if blood glucose levels are high. Levels are decreased with hemolysis, reducing accuracy as a marker of glycemia in sickle cell disease, thalassemia, and other chronic hemolytic anemias. (See 'Glycation/glycosylation' above and 'Role of Hb A1c in assessing blood glucose levels' above.)

ACKNOWLEDGMENT — 

UpToDate gratefully acknowledges Stanley L Schrier, MD, who contributed as Section Editor on earlier versions of this topic and was a founding Editor-in-Chief for UpToDate in Hematology.