HEMA1 - Hemoglobin Metabolism

Felix Seyler

In 1862, identified the respiratory protein hemoglobin, and discovered its characteristic colour spectrum, proving this was the true colouring matter of the blood.

Hemoglobin concentration

34 g/dL.

Hemoglobin MW

64,000 Daltons.

Free hemoglobin

Generated from RBCs through hemolysis. Has a short half-life.

X-ray crystallography

Hemoglobin was the first protein described using it.

Hemoglobin

Globular protein consisting of two different pairs of polypeptide chains and four heme groups.

Heme biosynthesis site

Occurs in the mitochondria and cytoplasm of bone marrow erythroid precursors — pronormoblasts through polychromatic erythrocyte. Predominantly produced in the red bone marrow and liver.

ALA synthase

Mitochondrion. Succinyl CoA + glycine » ALA.

ALA dehydratase

ALA » porphobilinogen. The step where the pathway crosses into the cytoplasm.

PBG deaminase

Cytoplasm. Porphobilinogen » hydroxymethylbilane.

Uroporphyrinogen III synthase

Cytoplasm. Hydroxymethylbilane » uroporphyrinogen III.

Uroporphyrinogen decarboxylase

Cytoplasm. Uroporphyrinogen III » coproporphyrinogen III.

Coproporphyrinogen III oxidase

Coproporphyrinogen III » protoporphyrinogen IX. The step where the pathway crosses back into the mitochondrion.

Protoporphyrinogen oxidase

Mitochondrion. Protoporphyrinogen IX » protoporphyrin IX.

Ferrochelatase

Mitochondrion. Protoporphyrin IX + Fe++ » HEME. The final step.

Heme pathway compartments

The pathway runs eight steps. It begins and ends in the mitochondrion and does its middle four steps in the cytoplasm.

Heme precursor mnemonic

While in the DELTA, POUR YOUR COP, PRONTO, a cup of HEME.
DELTA-aminolevulinic acid » PORphobilinogen » URoporphyrinogen » COProporphyrinogen » PROTOporphyrin » HEME + globin = hemoglobin

Globin chains

Two identical pairs of unlike polypeptide chains, 141 to 146 amino acids each. Variations in amino acid sequences give rise to the different types.

Helices

Rigid and linear. Designated A to H. Contain subgroup numberings for the sequence of the amino acids. Each chain is divided into 8 of them.

Nonhelical segments

Flexible; connect the helices. Designated NA, AB, BC, CD, DE, EF, FG, GH, HC. The deck states 7 segments and lists 9 designations.

Alpha chain

141 amino acids.

Beta chain

146 amino acids.

Gamma A chain

146 amino acids, with alanine at position 136.

Gamma G chain

146 amino acids, with glycine at position 136.

Delta chain

146 amino acids.

Epsilon chain

146 amino acids.

Zeta chain

141 amino acids.

Theta chain

Number of amino acids unknown. Appears only in the globin chains table, never in the slide text.

F8

The proximal histidine — the eighth residue of helix F, bonded directly to the heme iron.

E7

The distal histidine — the seventh residue of helix E, on the opposite face of the heme.

Heme pocket

The heme sits between F8 and E7, in the cleft the tertiary structure forms.

Globin structural genes

6 structural genes code for 6 globin chains.

Chromosome 16

Carries the alpha and zeta globin genes, on its short arm.

Chromosome 11

Carries the beta, gamma, delta and epsilon globin genes, on its short arm.

Globin gene copies

One copy of gene per chromatid, two genes per diploid cell. Exception: alpha and gamma contain 2 copies per chromatid, so four genes per diploid cell.

Alpha globin gene cluster

On chromosome 16, reads 5' to 3': Zeta 2 » Zeta 1 » Alpha 2 » Alpha 1 — two zeta genes and two alpha genes on one chromatid.

Globin synthesis steps

• Production
• Transcription to mRNA
• Translation of mRNA to the globin polypeptide chain

Globin regulation

Highly regulated for a balanced production of globin and heme. Controlled at the transcription level, by a complex interaction of DNA sequences and soluble transcription factors.

Promoter

The DNA sequences immediately before the 5' end of the gene. Required for initiation of transcription of a globin gene.

KLF1

Krüppel-like factor 1. Plays a key regulatory role in the switch from gamma chain to beta chain production, which begins in fetal life and continues through adulthood. Also regulates the expression of repressors of gamma globin gene transcription, such as BCL11A and MYB.

Other transcription factors

GATA1, Ikaros, TAL1, p45-NF-E2, LDB1.

Locus control region

LCR. An enhancer region of DNAse 1 hypersensitive nucleic acid sequences; the promoter for the beta-globin gene.

Primary structure

Amino acid sequence of the polypeptide chains.

Secondary structure

Chain arrangements in helices and nonhelices.

Tertiary structure

Arrangement of the helices into a pretzel-like configuration. The globin chains loop, forming a cleft pocket for heme. Amino acids in the cleft are hydrophobic; those on the outside are hydrophilic.

Quaternary structure

Spherical. Has 4 heme groups attached to 4 polypeptide chains, and carries 4 molecules of oxygen.

Heterodimer

Formed by the binding of a globin chain to a heme molecule.

Alpha chain affinity

The alpha chain has a positive charge, and the highest affinity for a beta chain (negative charge). Next highest is the gamma and delta globin chain.

Hemoglobin assembly

Two heterodimers combine and complete the hemoglobin molecule.

Hemoglobin ontogeny

Hemoglobin changes reflect the sequential activation and inactivation of the globin genes. Three groups: embryonic, fetal and hemoglobin A.

Embryonic hemoglobin

Formed in the yolk sac. Predominant hemoglobin during the 1st trimester.

Gower I

2 zeta + 2 epsilon. An embryonic hemoglobin.

Gower II

2 alpha + 2 epsilon. An embryonic hemoglobin.

Portland

2 zeta + 2 gamma. An embryonic hemoglobin.

Hemoglobin F

2 alpha + 2 gamma. Predominant during the 2nd and 3rd trimesters of fetal life and at birth.

Hemoglobin A

2 alpha + 2 beta. Predominant by 6 months of age and through adulthood.

Hemoglobin A2

2 alpha + 2 delta. Increases during the first year of life until the adult level is reached. Comprises < 3.5 % in adults.

Glycation

A post-translational modification formed by the nonenzymatic binding of various sugars to globin chain amino groups over the life span of the RBC.

Hemoglobin A1

A subfraction of hemoglobin A. The glycosylated hemoglobin.

HbA1c

The most characterized glycated hemoglobin. Glucose attaches to the N-terminal valine of the beta chain. It is 4 % to 6 % of total hemoglobin in adults.

Hemoglobin at birth

F, 60 %–90 %.
A, 10 %–40 %.

Hemoglobin in adulthood

Two years through adulthood:
F, 1 %–2 %
A2, < 3.5 %
A, > 95 %

Hemoglobin F timing

Begins in early embryogenesis; peaks during the third trimester and begins to decline just before birth.

Hypoxia

Occurs when there is an insufficient quantity of hemoglobin, or if the hemoglobin molecule is defective in transporting oxygen. Detected by the peritubular cells of the kidneys, which respond by increasing EPO.

Hemoglobin functions

• Oxygen transport
• Carbon dioxide transport
• Nitric oxide transport

Oxygen affinity requirement

High oxygen affinity to transport oxygen, and low oxygen affinity to efficiently unload it to the tissues.

Oxygen binding capacity

Each of the 4 heme iron atoms can reversibly bind 1 oxygen molecule.

Oxygen bound per gram

1.34 mL of oxygen is bound by each gram of hemoglobin.

Oxygen dissociation curve

Plots the percent oxygen saturation of hemoglobin against the PO2. The curve is sigmoidal.

PO2

Related to the affinity of hemoglobin for oxygen.

P50

The amount of oxygen needed to saturate 50 % of hemoglobin. 27 mm Hg PO2 results in 50 % oxygen saturation of the hemoglobin molecule.

Shift to the left

Occurs at a PO2 of < 27 mm Hg.

Shift to the right

Occurs at a PO2 of > 27 mm Hg.

Myoglobin

Plotted alongside hemoglobin on the dissociation curve, and its curve is not sigmoidal — it saturates far faster at low oxygen tension. Appears only in the figure, never in the slide text.

T state

Tense conformation. The hemoglobin tetramer is deoxygenated. Stabilized by the binding of 2,3-BPG between the beta-globin chains. Shifts to the RIGHT with the binding of 2,3-BPG, lower pH, and higher PCO2.

R state

Relaxed state. The hemoglobin tetramer is fully oxygenated. With oxygen uptake in the lungs the salt bonds are sequentially broken, the beta chains are pulled together, expelling 2,3-DPG.

Carbonic anhydrase

An RBC enzyme that facilitates the reaction in venous blood, the first step of carbon dioxide transport.

Carbonic acid

Dissociates to release H+ and bicarbonate.

Bohr effect

H+ binds oxygenated hemoglobin (HbO2), and the oxygen then diffuses out of the cell into the tissues.

Chloride shift

As the concentration of the negatively charged bicarbonate increases it diffuses across the RBC membrane into the plasma. To maintain electroneutrality, chloride diffuses from the plasma into the cell.

CO2 in the lungs

Bicarbonate is carried back to the lungs by the plasma. In the pulmonary capillaries it is converted back into carbon dioxide and water and eliminated through respiration.

Carbamino group

A small percentage of CO2 remains in the cytoplasm, and the remainder binds to the globin chains as this.

Nitric oxide

Secreted by vascular endothelial cells. Causes relaxation of the vascular wall smooth muscle and vasodilation.

S-nitrosohemoglobin

Formed when free nitric oxide enters the RBCs and binds to cysteine in the beta chain of hemoglobin.

Methemoglobin

Formed by the reversible oxidation of heme iron to the ferric state.

NADH-cytochrome b5 reductase 3

Also called NADH-methemoglobin reductase. Limits methemoglobin to 1 % during normal oxygenation and deoxygenation.

Methemoglobin levels

• < 25 % — asymptomatic
• > 30 % — cyanosis and hypoxia (dyspnea, headache, vertigo, change in mental status)
• > 50 % — can lead to coma and death

Methemoglobinemia

An increase in the methemoglobin level that results in decreased delivery of oxygen to the tissues. Can be acquired or hereditary. Infants are more susceptible.

Acquired methemoglobinemia

Exposure to an exogenous oxidant: nitrites, primaquine, dapsone, benzocaine.

Acquired methemoglobinemia

Treatment: withdrawal of agent, intravenous methylene blue, exchange transfusion.

Hereditary methemoglobinemia

Mutations in the gene for NADH-cytochrome b5 reductase 3 (CYB5R3), which result in a diminished capacity to reduce methemoglobin; and mutations in the alpha, beta, gamma-globin gene, which result in binding of the ferric form and prevent reduction.

M hemoglobin

Hb M. Inherited in an autosomal dominant pattern, with methemoglobin comprising 30 % to 50 % of total hemoglobin.

Cytochrome b5 reductase deficiency

An autosomal recessive disorder. Elevations occur in individuals who are homozygous or compound heterozygous for a CYB5R3 mutation.

MetHb assay

Spectral absorption analysis — CO oximeter. Absorption peak 630 nm.

MetHb colour

Chocolate brown, and it does not revert back to the normal red colour after oxygen exposure.

Hb M testing

Hemoglobin electrophoresis, HPLC, DNA mutation testing.

Sulfhemoglobin

Formed by the irreversible oxidation of hemoglobin by drugs or exposure to sulfur chemicals in industrial or environmental settings. Addition of a sulfur atom to the pyrrole ring of heme.

Sulfhemoglobin drugs

Sulfanilamides, phenacetin, nitrites and phenylhydrazine.

Sulfhemoglobin colour

Greenish pigment.

Sulfhemoglobin clinical

Presents with cyanosis. Cannot be converted to normal Hb A. Treatment: avoidance of the offending agent.