Hemoglobin Metabolism

Mary Rose M. Apuyan, RMT, DTA

 Hemoglobin Structure
● First protein whose structure was described using x-ray
crystallography.
● The hemoglobin molecule is a globular protein consisting of
two different pairs of polypeptide chains and four heme
groups, with one heme group imbedded in each of the four
polypeptide chains.
Hemoglobin is an oxygen-transporting
protein contained within erythrocytes.

The heme portion of hemoglobin gives

erythrocytes their characteristic red
color.
 Heme Structure
• Consists of a ring of carbon, hydrogen, and nitrogen atoms called
protoporphyrin IX, with a central atom of divalent ferrous iron
(Fe2+).
• Each of the four heme groups is positioned in a pocket of the
polypeptide chain near the surface of the hemoglobin molecule.
• The ferrous iron in each heme molecule reversibly combines with
one oxygen molecule.
• When the ferrous irons are oxidized to the ferric state (Fe 3+), they no
longer can bind oxygen.
• Methemoglobin = oxidized hemoglobin
 Globin Structure
• The four globin chains comprising
each hemoglobin molecule consist
of two identical pairs of unlike
polypeptide chains, 141 to 146
amino acids each.
• Variations in amino acid sequences
give rise to different types of
polypeptide chains.
• Each chain is designated by a
Greek letter.
 Globin Structure
• Each globin chain is divided into eight helices separated by
seven non-helical segments.
• The helices, designated A to H, contain subgroup numberings for the
sequence of the amino acids in each helix and are relatively rigid
and linear.
• Flexible nonhelical segments connect the helices, as reflected
by their designations: NA for the sequence between
• the N-terminus and the A helix, AB between the A and B helices, and
so forth, with BC, CD, DE, EF, FG, GH, and finally HC between the
H helix and the C-terminus.
 Hemoglobin Molecule
• Primary structure- amino acid sequence of the polypeptide chains.
• Secondary structure - chain arrangements in helices and non-
helices.
• Tertiary structure - arrangement of the helices into a pretzel-like
configuration.
Globin chains loop to form a cleft pocket for heme.
Each chain contains a heme group that is suspended between the E and
F helices of the polypeptide chain.
 Hemoglobin Molecule
Globin chain amino acids in the cleft are hydrophobic, whereas amino acids
on the outside are hydrophilic, which renders the molecule water soluble.
This arrangement also helps iron remain in its divalent ferrous form regardless of
whether it is oxygenated (carrying an oxygen molecule) or deoxygenated (not
carrying an oxygen molecule).

• Quaternary structure - a tetramer, the complete hemoglobin molecule.

The complete hemoglobin molecule is spherical, has four heme groups
attached to four polypeptide chains, and may carry up to four molecules of
oxygen.
 Hemoglobin Biosynthesis
• 65% hemoglobin synthesis occurs in immature nRBCs.
• 35% hemoglobin synthesis occurs in reticulocytes.
• Heme synthesis occurs in the mitochondria of normoblasts and is
dependent on glycine, succinyl coenzyme A, aminolevulinic acid
synthetase, and vitamin B6 (pyridoxine).
• Globin synthesis occurs in the ribosomes, and it is controlled on
chromosome 16 for alpha chains and chromosome 11 for all other
chains.
• Each globin chain binds to a heme molecule in the cytoplasm of the
immature RBC.
 Heme Synthesis
• Begins in the mitochondria with the condensation of glycine and
succinyl coenzyme A (CoA) catalyzed by aminolevulinate synthase
to form aminolevulinic acid (ALA).
• In the cytoplasm, aminolevulinic acid dehydratase (also known as
porphobilinogen synthase) converts ALA to porphobilinogen (PBG).
• PBG undergoes several transformations in the cytoplasm from
hydroxylmethylbilane to coproporphyrinogen III.
• This pathway then continues in the mitochondria until, in the final step of
production of heme, Fe2+ combines with protoporphyrin IX in the
presence of ferrochelatase (heme synthase) to make heme.
 Heme Synthesis
• Transferrin - a plasma protein, carries iron in the ferric form to
developing erythroid cells; binds to transferrin receptors on erythroid
precursor cell membranes and the receptors and transferrin (with bound
iron) are brought into the cell in an endosome.
• Acidification of the endosome releases the iron from transferrin.
• Iron is transported out of the endosome and into the mitochondria
where it is reduced to the ferrous state, and is united with
protoporphyrin IX to make heme.
• Heme leaves the mitochondria and is joined to the globin chains in the
cytoplasm.
 Globin Synthesis
• The production of globin chains takes place in erythroid
precursors from the pronormoblast through the circulating
polychromatic erythrocyte, but not in the mature erythrocyte.

• Transcription of the globin genes to messenger ribonucleic acid

(mRNA) occurs in the nucleus, and translation of mRNA to the globin
polypeptide chain occurs on ribosomes in the cytoplasm.

• After translation is complete, the chains are released from the

ribosomes in the cytoplasm.
 Hemoglobin Ontogeny
• The z- and e-globin chains normally appear only during the first 3
months of embryonic development.
• These two chains, when paired with the a and g chains, form the
embryonic hemoglobins.
• During the second and third trimesters of fetal life and at birth, Hb F
(a2g2) is the predominant hemoglobin.
• By 6 months of age and through adulthood, Hb A (a2b2) is the
predominant hemoglobin, with small amounts of Hb A2 (a2d2) and
Hb F.
 Types of Hemoglobin
• Hgb F contains two alpha- and two gamma-globin chains.
• Hgb F functions in a reduced oxygen environment.
• Hgb F predominates at birth (80%). Gamma chain production switches
over to beta chain production and is complete by 6 months of age.
a. Laboratory: Alkali denaturation test and Kleihauer-Betke acid
elution
stain (Hgb F is resistant to denaturation/elution), column
chromatography, radial immunodiffusion
b. Hgb F is a compensatory hemoglobin and can be increased in
homozygous hemoglobinopathies and beta-thalassemia major.
 Types of Hemoglobin
• Adult
a. Hgb A contains two alpha- and two beta-globin chains.
1) Hgb A is subdivided into glycosylated fractions.
2) Alc fraction reflects glucose levels in the blood and is used to
monitor individuals with diabetes mellitus.
b. Hgb A2 contains two alpha- and two delta-globin chains.
c. Reference range for a normal adult is 97% Hb A, 2% Hb A2, and 1%
Hb F.
 Hemoglobin/Erythrocyte Breakdown
1. Intravascular hemolysis (10%)
a. Occurs when hemoglobin breaks down in the blood and free
hemoglobin is released into plasma
b. Free hemoglobin binds to haptoglobin (major free hemoglobin
transport protein), hemopexin, and albumin, and it is phagocytized
by liver macrophages.
c. Laboratory: Increased plasma hemoglobin, serum bilirubin,
serum LD, and urine urobilinogen; hemoglobinuria and
hemosiderinuria present; decreased serum haptoglobin
 Hemoglobin/Erythrocyte Breakdown
2. Extravascular hemolysis (90%)
a. Occurs when senescent/old RBCs are phagocytized by
macrophages in the liver or spleen
b. Protoporphyrin ring metabolized to bilirubin and urobilinogen;
excreted in urine and feces
c. Globin chains are recycled into the amino acid pool for protein
synthesis.
d. Iron binds to transferrin and is transported to bone marrow for new
RBC production, or it is stored for future use in the form of ferritin or
hemosiderin.
 Hemoglobin and Iron
1. Most iron in the body is in hemoglobin and must be in the ferrous
state to be used. Fe2+ binds to oxygen for transport to lungs and
body tissues.
2. Ferric iron (Fe3+) is not able to bind to hemoglobin, but does bind to
transferrin. Iron is an essential mineral and is not produced by the body.
a. Serum iron measures the amount of Fe3+ bound to transferrin.
b. Total iron-binding capacity (TIBC) measures the total amount of iron
that transferrin can bind when fully saturated.
c. Serum ferritin is an indirect measurement of storage iron in tissues and
bone marrow.
 Different Forms of Normal Hemoglobin
• Oxyhemoglobin: Hemoglobin with Fe2+ + O2; seen in arterial circulation
• Deoxyhemoglobin: Hemoglobin with Fe2+ but no O2; seen in venous
circulation
• Carboxyhemoglobin: Hemoglobin with Fe2+ and carbon monoxide (CO);
hemoglobin has 200 X more affinity for CO than O2 so CO is earned
instead of O2; can result in death, but is reversible if given pure O2
• Sulfhemoglobin: Hemoglobin with S; cannot transport O2; seldom
reaches fatal levels; caused by drugs and chemicals; irreversible, not
measured by the cyanmethemoglobin method
• Methemoglobin: Hemoglobin with Fe3+; cannot transport O2; increased
• levels cause cyanosis and anemia
 Oxygen Dissociation Curve
1. Oxygen affinity is the ability of hemoglobin to bind or
release oxygen. Expressed in terms of the oxygen
tension at which hemoglobin is 50% saturated with
oxygen.

2. The relationship between oxygen tension and hemoglobin

saturation with oxygen is described by the oxygen
dissociation curve.
 Oxygen Dissociation Curve
a. Right shift decreases oxygen affinity, more O2 release
to the tissues— high 2,3-bisphosphoglycerate (formerly
2,3-diphosphoglycerate/2,3-DPG) level or increased body
temperature; decreased body pH

b. Left shift increases oxygen affinity, less O2 release to

the tissues—low 2,3-bisphosphoglycerate (2,3-BPG) level or
decreased body temperature; increased body pH