Hemoglobin

Supachai A. Basit
HEMOGLOBIN

 The name hemoglobin is the concatenation

of heme and globin, reflecting the fact that
each subunit of hemoglobin is a globular
protein with an embedded heme (or haem)
group;
 Each heme group contains an iron atom,
and this is responsible for the binding of
oxygen.
 The most common types of hemoglobin
contains four such subunits, each with one
heme group.
 Biosynthesis of Heme
Succinyl coenzyme A + Glycine

Pyridoxal PO4 delta aminolevulinic acid syn

delta aminolevulinic acid

DAA dehydrase
porphobilinogen
uroporphorinogen III cosyn
 Uroporphorynogen
Uroporphorynogen decarboxylase
Coproporphyrinogen III
Coprophorphyrinogen oxidase
Protoporphyrinogen IX
Protoporphyrinogen oxidase
Protoporphyrin IX
Ferrochetalase Fe+

Heme
In summary….
Heme

 A heme group consists of an iron

atom held in a heterocyclic ring,
known as a porphyrin.
 This iron atom is the site of oxygen
binding. The iron atom is bonded
equally to all four nitrogens in the
center of the ring, which lie in one
plane.
 Heme
 Two additional bonds perpendicular to the
plane on each side can be formed with the
iron to a fifth and sixth bonding position, one
connected strongly to the protein, the other
available for binding of an oxygen molecule.
 The iron atom may either be in the Fe2+ or
Fe3+ state, but ferrihemoglobin
(methemoglobin)
 (Fe3+) cannot bind oxygen.
 Blood with high carbon dioxide levels
is also lower in pH (more acidic).
 Hemoglobin can bind protons and
carbon dioxide which causes a
conformational change in the protein
and facilitates the release of oxygen.
 Protons bind at various places along
the protein, and carbon dioxide binds
at the alpha-amino group forming
carbamate.
 Conversely, when the carbon dioxide levels
in the blood decrease (i.e., in the lung
capillaries), carbon dioxide and protons are
released from hemoglobin, increasing the
oxygen affinity of the protein.
 This control of hemoglobin's affinity for
oxygen by the binding and release of
carbon dioxide and acid, is known as the
Bohr effect.
Differentiation of Hb

 Embryonic
 Fetal

 Human
Embryonic

 Gower 1 (ξ2ε2)
 Gower 2 (α2ε2)
 Hemoglobin
Portland (ξ2γ2)
Fetal Hb

 Hemoglobin F.
Hemoglobin F is
the predominant
hemoglobin during
fetal development.
T
 The molecule is a
tetramer of two
alpha chains and
two gamma chains
(a2g2).
Fetal Hb

 The genes for hemoglobin F and

hemoglobin A are closely related, existing in
the same gene cluster on chromosome 11.
 Hemoglobin F production falls dramatically
after birth, although some people continue
to produce small amounts of hemoglobin F
for their entire lives.
 Alkali denaturation test (HbF)
 Acid elution test (Kleihauer-Betke Test)
HbF
Adult Human Hb

 Hemoglobin A1 (α2β2) - The most

common type.
 Hemaglobin A2 (α2δ2) - δ chain
synthesis begins late in the third
trimester and in adults, it has a normal
level of 2.5%
 Hemoglobin F (α2γ2) - In adults
Hemoglobin F is restricted to a limited
population of red cells called F cells.
Adult Human

 In adult humans, the most common

hemoglobin type is a tetramer (which
contains 4 subunit proteins) called
hemoglobin A, consisting of two α and two
β subunits non-covalently bound
 Each made of 141 and 146 amino acid
residues, respectively. This is denoted as
α2β2.
 The subunits are structurally similar and
about the same size
Adult Human

 Each subunit has a molecular weight

of about 16,000 daltons, for a total
molecular weight of the tetramer of
about 64,000 daltons.
 Hemoglobin A is the most intensively
studied of the hemoglobin molecules.
In summary…
Normal Hb

1. HbO2 = bright red

2. Reduced/Deoxygenated= HbCO2
Very important fact!!!
Abnormal Hb

 Hi
 HbCO

 SHb
Hi

 The iron atom in the heme group must be

in the Fe2+ oxidation state to support
oxygen and other gases' binding and
transport.
 Oxidation to Fe3+ state converts
hemoglobin into hemiglobin which cannot
bind oxygen.
 Hemoglobin in normal red blood cells is
protected by a reduction system to keep
this from happening.
Hi

 1.5 gm/ml
 Evelyn Malloy

 chocolate brown
 HbCO
 The binding of oxygen is affected by
molecules such as carbon monoxide (CO)
(for example from tobacco smoking, cars
and furnaces).
 CO competes with oxygen at the heme
binding site.
 Hemoglobin binding affinity for CO is 200
times greater than its affinity for oxygen,
meaning that small amounts of CO
dramatically reduces hemoglobin's ability
to transport oxygen.
HbCO

 irreversible
 undissociable; cherry red

 5gm/100ml

 Sunderman dithionate spectroscopic

method
 Katayama’s test
 When hemoglobin combines with CO, it
forms a very bright red compound called
carboxyhemoglobin.
 When inspired air contains CO levels as low
as 0.02%, headache and nausea occur; if
the CO concentration is increased to 0.1%,
unconsciousness will follow. In heavy
smokers, up to 20% of the oxygen-active
sites can be blocked by CO.
HbCO
SHb

 mauve lavander
 0.5 gm/100 ml

 irreversible
Different pigments of hb
HbA1c

 This Hb A1c level is only useful in

individuals who have red blood cells
(RBCs) with normal survivals (i.e.,
normal half-life).
HbA1C
Hemoglobinopathy
 Genetic defect that results in abnormal
structure of one of the globin chains of the
hemoglobin molecule.
 Although the suffix "-pathy" would conjure
an image of "disease," most of the
hemoglobinopathies are not clinically
apparent.
 Others produce asymptomatic abnormal
hematologic laboratory findings. A very few
produce serious disease.
 The genetic defect may be due to
substitution of one amino acid for another
 Pattern of hemoglobin electrophoresis from several
different individuals. Lanes 1 and 5 are hemoglobin
standards. Lane 2 is a normal adult. Lane 3 is a
normal neonate. Lane 4 is a homozygous HbS
individual. Lanes 6 and 8 are heterozygous sickle
individuals. Lane 7 is a SC disease individual.
 Hb S Beta 6th glu  val
 HbC Beta 6th glu  lys
 HbC Chesapeake Alpha 92nd arg  leu
 Hb Rainier Beta 145 tyr  cys
 Hb Hiroshima Beta 146th his  asp
 Hb Tacoma Beta 30th argser
 Hb Zurich Beta 63rd his  arg
 Hb Gunhill deletion beta 93rd-97th
leu
 Hb Kansas Beta 102nd asnthr
 Hb Hammersmith 42nd pheser
 Hb Seattle Beta 76th alaglu
 Hb Torino alpha 43rd pheval
 Hb M Boston alpha 58th histyr
 Hb M Saskatoon beta 63rd his tyr
 Hb M Hyde Park beta 92nd his tyr
 Hb Geneva beta 28th leu 
pro
HbS

 This the predominant hemoglobin in

people with sickle cell disease. The
alpha chain is normal.
 The disease-producing mutation
exists in the beta chain, giving the
molecule the structure, a2bS2.
 People who have one sickle mutant
gene and one normal beta gene have
sickle cell trait which is benign.
HbC

 Hemoglobin C results from a mutation

in the beta globin gene and is the
predominant hemoglobin found in
people with hemoglobin C disease
(a2bC2).
 Hemoglobin C disease is relatively
benign, producing a mild hemolytic
anemia and splenomegaly.
Hemoglobin C trait is benign.
Hb C Hb S
HbE

 This variant results from a mutation in

the hemoglobin beta chain.
 People with hemoglobin E disease
have a mild hemolytic anemia and
mild splenomegaly.
 Hemoglobin E trait is benign.
 Hemoglobin E is extremely common
in S.E. Asia and in some areas equals
hemoglobin A in frequency.
 Hb Constant Spring
 a variant in which a mutation in the alpha globin gene
produces an alpha globin chain that is abnormally
long.
 The quantity of hemoglobin in the cells is low for two
reasons.
 First, the messenger RNA for hemoglobin Constant
Spring is unstable.
 Some is degraded prior to protein synthesis.
 Second, the Constant Spring alpha chain protein is
itself unstable. The result is a thalassemic phenotype.
Hb H

 a tetramer composed of four beta

globin chains.
 Hemoglobin H occurs only with
extreme limitation of alpha chain
availability.
 Hemoglobin H forms in people with
three-gene alpha thalassemia as well
as in people with the combination of
two-gene deletion alpha thalassemia
and hemoglobin Constant Spring.
Hb Bart

 develops in fetuses with four-gene

deletion alpha thalassemia.
 During normal embryonic
development, the episilon gene of the
alpha globin gene locus combines
with genes from the beta globin locus
to form functional hemoglobin
molecules.
 The episolon gene turns off at about 12
weeks, and normally the alpha gene takes
over.
 With four-gene deletion alpha thalassemia
no alpha chain is produced.
 The gamma chains produced during fetal
development combine to form gamma chain
tetramers.
 These molecules transport oxygen poorly.
Most individuals with four-gene deletion
thalassemia and consequent hemoglobin
Barts die in utero (hydrops fetalis
Hemoglobinometry
1. Visual Colorimetry
a. Acid Hematin (sahli adam/sahli hellige)
b. Alkali Hematin
c. Tallquist
d. Dares
2. Photoelectric Colorimetry
a. HiCN
b. HbO2
3. Specific Gravity
4. Gasometry
5. Chemical Method
 Complete Blood Count

SAHLI’S ACID HAEMATIN METHOD

HEMOGLOBIN
 less accurate because the colour
develops slowly, is unstable, and
begins to fade almost immediately
after it reaches its peak.
 Gives a true estimate of total Hb
 plasma proteins and lipids have
little effect on the development of
colour, although they cause
turbidity.
Hemometer Set
 Complete Blood Count

 A conjugated protein that

HEMOGLOBIN
serves as the vehicle for
transportation of oxygen &
carbon dioxide.
SPECTROPHOTOMETER

PHOTOELECTRIC
COLORIMETER

Two methods are in common use:

 (a) Cyanmethemoglobin method
 (b) Oxyhemoglobin method.
Hb K3Fe(CN)6 Hi (unstable)
NaHCO3
KCN

HiCN 540 nm
HiCN

 the best method

 Drabkin’s reagents

 540 nm
 Complete Blood Count

CYANMETHEMOGLOBIN METHOD
HEMOGLOBIN
 Recommended method
 Basis: dilution of blood in a
solution containing potassium
cyanide and potassium
ferricyanide.
 Haemoglobin, Hi, and HbCO, but
not SHb, are converted to HiCN.
 Absorbance: 540 nm

This slide is courtesy of Ms. Lea Ballares

 Complete Blood Count

CYANMETHEMOGLOBIN METHOD
HEMOGLOBIN
DILUENT: DRABKIN’S REAGENT
(pH of 8:6)
MODIFIED DRABKIN’S REAGENT
(pH of 7.0-7.4)
 Less likely to cause turbidity from
precipitation of plasma proteins
 Requires shorter conversion time (3-5
minutes)
 DISADVANTAGE: Detergent causes
frothing
This slide is courtesy of Ms. Lea Ballares
 Complete Blood Count

CYANMETHEMOGLOBIN METHOD
HEMOGLOBIN
MODIFIED DRABKIN’S REAGENT
(pH of 7.0-7.4)

Potassium ferricyanide (0.607 mmol/l) 200 mg

Potassium cyanide (0.768 mmol/l) 50 mg

Potassium dihydrogen phosphate 140 mg

(1.029 mmol/l)
Nonionic detergent 1 ml
Distilled or deionized water To 1 litre

This slide is courtesy of Ms. Lea Ballares

 Creating a hemoglobin
standard curve
Hb concentration (g/L) Blank 5 10 15 20

Volume of HiCN standard (mL) 0 1.5 3 4.5 6

Volume of HiCN Rgt (Drabkin’s) 6 4.5 3 1.5 0

ml

Plot the standard calibration curve when a standard of 80 mg/dL is used.

Blank: 100%T
5 g/dL: 72.9%T
10 g/dL: 53.2%
15 g/dL: 39.1%T
20 g/dL: 28.7%
 You can also compute for Hb
concentration using the ff formula

Hb Conc. = OD of unknown x conc of std

OD of standard
 Complete Blood Count

OXYHEMOGLOBIN METHOD
HEMOGLOBIN
 Simplest and quickest method for
general use with a photometer.

 Disadvantage: It is not possible to

prepare a stable HbO2 standard,
so the calibration of these
instruments should be checked
regularly using HiCN reference
solutions.

This slide is courtesy of Ms. Lea Ballares

 Complete Blood Count

OXYHEMOGLOBIN METHOD
HEMOGLOBIN
 Wash 20 ml of blood into a tube
containing 4 ml of 0.4 ml/l ammonia
(specific gravity 0.88) to give a ×201
dilution.
 Use a tightly fitting stopper and mix by
inverting the tube several times. The
solution of HbO2 is then ready for
matching against a standard in a
spectrometer at 540 nm or a photometer
with a yellow–green filter (e.g., Ilford
625) against a water blank.

This slide is courtesy of Ms. Lea Ballares

 Complete Blood Count

OXYHEMOGLOBIN METHOD
HEMOGLOBIN
 If the absorbance of the haemoglobin
solution exceeds 0.7, dilute the blood
further with an equal volume of water,
and read again.
Specific Gravity Method
 Copper sulfate
method
 Based on the
principle that a
normal blood has
a specific gravity
of 1.055
 For screening of
blood donors
 NORMAL HEMOGLOBIN
AGE AND SEX
LEVEL (g/dL)

Children: 6 mos - 6y
Normal Hemoglobin Levels Based on WHO Cut-Off, 1972 11.0
6.1y - 14y 12.0

Adults: Males 14-18

Females (Non- 12-15
pregnant/non-
lactating)
Pregnant Women 11.0
Lactating Women 12.0
Conversion Factor
 Hb% x standard = Hb in gm/100 ml
100

Hb gm/100 ml x 10 = g/L

Hb gm/100 ml x 100 = Hb%

STD

Hb in g/L = Hb in gm/100 ml
10