Structure, function and metabolism

of hemoglobin

Pavla Balínová

Figure was adopted from http://themedicalbiochemistrypage.org/hemoglobin-myoglobin.html

 Hemoglobin (Hb)

is a hemoprotein only found in the cytoplasm of
erythrocytes (ery)
 transports O and CO between lungs and various
2 2
tissues

normal concentration of Hb in the blood:
adult males 135 – 175 g/L
adult females 120 – 168 g/L

Data were assumed from Department of biochemistry and pathobiochemistry FNKV

 Structure of hemoglobin
• Hb is a spherical molecule consisting of 4 peptide subunits
(globins) = quartenary structure
• Hb of adults (Hb A) is a tetramer consisting of 2 - and 2 β-
globins → each globin contains 1 heme group with a central
Fe2+ ion (ferrous ion)

Figure is found at http://faculty.etsu.edu/currie/images/hemat3.jpg

 Heme structure
Heme is a metaloporphyrine
(cyclic tetrapyrrole)
Heme contains:
 conjugated system of
double bonds → red colour
 4 nitrogen (N) atoms

 1 iron cation (Fe2+)

→ bound in the middle of
tetrapyrrole skelet by
coordination covalent
bonds

methine bridge pyrrole ring

Figure is found at http://www.nebrwesleyan.edu/people/nfackler/lecturelinks/images/

Overheads/Fig%2015-A.jpg
 Properties of iron in heme

• Coordination number of
iron in heme = 6

6 bonds:
• 4x pyrrole ring (A,B,C,D)
• 1x link to a protein
• 1x link to an oxygen

Figure is found at http://www.mun.ca/biochem/courses/3107/images/VVP/Ch07/7-2.jpg

In which compounds can we find a heme
group?
Hemoproteins
• Hemoglobin (Hb)
• Myoglobin (Mb)
• Cytochromes
• Catalases (decomposition
of 2 H2O2 to 2 H2O and O2)
• Peroxidases

Figure was assumed from http://en.wikipedia.org/wiki/Heme

 Myoglobin (Mb)

• is a single-chain globular
protein of 153 AA,
containing 1 heme group
• transports O2 in skeletal
and heart muscle
• is found in cytosol
within cells
• is a marker of myocard
damage

Figure is found at http://en.wikipedia.org/wiki/Myoglobin

 Heme biosynthesis - repetition
• in bone marrow (85% of Hb) and liver (cytochromes)
• cell location: mitochondria / cytoplasm / mitochondria
• substrates: succinyl-CoA + glycine
• important intermediates:
 -aminolevulinic acid (= 5-aminolevulinic acid, ALA)
 porphobilinogen (PBG = pyrrole derivate)
 uroporphyrinogen III (= porphyrinogen – heme
precursor)
 protoporphyrin IX (= direct heme precursor)
● key regulatory enzyme: ALA synthase
 δ-aminolevulinic acid (ALA)
• synthesis of heme starts in mitochondria
• succinyl-CoA and Gly undergo a condensation → ALA
• reaction is catalyzed by enzyme ALA synthase

Figure was assumed from textbook Harper´s Illustrated Biochemistry 26th ed./
R.K.Murray; McGraw-Hill Companies, 2003, ISBN 0-07-138901-6.
 Porphobilinogen (PBG)
• ALA leaves the mitochondria → cytoplasm
• 2x ALA condense together to form porphobilinogen
• reaction is catalyzed by porphobilinogen synthase
(ALA dehydratase)

Figure was assumed from textbook Harper´s Illustrated Biochemistry 26th ed./
R.K.Murray; McGraw-Hill Companies, 2003, ISBN 0-07-138901-6.
 Uroporphyrinogen → coproporphyrinogen III
• enzyme hydroxymethylbilane synthase catalyzes the linkage of
four PBG molecules to yield uroporphyrinogen III
• 4 acetate residues are decarboxylated into methyl groups →
coproporphyrinogen III returns to the mitochondria again

Figure was assumed from book T. M. Devlin et al.: Textbook of Biochemistry With Clinical
Correlations, 4th ed., Wiley‑Liss, Inc., New York, 1997.
 Protoporphyrinogen IX → protoporphyrin IX

• oxidation of protoporphyrinogen IX produces the

conjugated π-electrone system of protoporphyrin IX

Figure was assumed from book T. M. Devlin et al.: Textbook of Biochemistry With Clinical
Correlations, 4th ed., Wiley‑Liss, Inc., New York, 1997.
 Final formation of heme

• Fe2+ is incorporated into protoporphyrin IX

• reaction is catalyzed by enzyme ferrochelatase

Figure was assumed from http://www.porphyrin.net/mediporph/_netbiochem/synthesis/

ferrochelatase.html
 Regulation of heme biosynthesis
 ALA synthase is a key regulatory enzyme
●it is an allosteric enzyme that is inhibited by an end product - heme
(feedback inhibition)
●requires pyridoxal phosphate as a coenzyme
●certain drugs and steroid hormones can increase heme synthesis

 Porphobilinogen synthase is inhibited by lead ions Pb2+ in case of lead

poisoning.

 Ferrochelatase (heme synthase) can be also inhibited by Pb2+. Its

activity is influenced by availability of Fe2+ and ascorbic acid.
Figure was assumed from book Color Atlas of Biochemistry / J. Koolman, K.H.Röhm. Thieme 2005.
ISBN 3-13-100372-3
 Hemoglobin degradation
In the human body approx. 100 – 200 million ery are
broken down every hour. Degradation of Hb begins in ER
of reticuloendothelial cells (RES) of the liver, spleen,
bone marrow and skin.

Hb is degraded to:
● globins → AAs → metabolism
● heme → bilirubin
● Fe2+ → transported with transferrin and used in the
next heme biosynthesis

Not only Hb but other hemoproteins also contain heme

groups which are degraded by the same pathway.
 Convertion of heme to bilirubin

green

red-orange

Figure is found at http://themedicalbiochemistrypage.org/heme-porphyrin.html

 Futher fate of bilirubin
Bilirubin (Bil) is released from RES into the blood. BUT!
Bil is only poorly soluble in plasma, and therefore during
transport it is bound to albumin („nonconjugated Bil“).
↓

LIVER
In the hepatocytes, Bil is conjugated by 2 molecules of
glucuronic acid → bilirubin diglucuronide (soluble in
water, „conjugated Bil“)
↓
BILE
↓
INTESTINE
Bil is reduced to urobilinogen and stercobilinogen
 bilirubin-diglucuronide = conjugated bilirubin
is soluble in water → „direct bilirubin“

Figure is found at http://themedicalbiochemistrypage.org/heme-porphyrin.html

 In intestine:
Urobilinogen and stercobilinogen can be:
a) reabsorbed and returned to the liver
(= enterohepatic circulation)

b) oxidized (in the presence of O2) to pigments urobilin

(orange) and stercobilin (yellow) → they are excreted
in the stool

Urobilinogen also appears in the urine.

 Bile pigments:
• bilirubin
• urobilin
• stercobilin

Figure was assumed from book Color Atlas of Biochemistry / J. Koolman, K.H.Röhm. Thieme 2005.
 Clinical correlations
Determination of bilirubin (Bil) in serum
Blood tests
 Bil reacts directly when reagents are added to the blood
sample → conjugated bilirubin = direct Bil (up to 3.4 µmol/L)

 free Bil does not react to the reagents until alcohol

(methanol) or caffeine is added to the solution. Therefore,
the measurement of this type of bilirubin is indirect →
unconjugated bilirubin = indirect Bil (up to 13.6 µmol/L)

 total bilirubin measures both unconjugated and conjugated

Bil (normal value up to 17 µmol/L).
 Types of hemoglobin
 Adult Hb (Hb A) = 2 α and 2 β subunits
HbA1 is the major form of Hb in adults and in children over 7 months.
HbA2 (2 α, 2 δ) is a minor form of Hb in adults. It forms only 2 – 3% of
a total Hb A.

 Fetal Hb (Hb F) = 2 α and 2 γ subunits

- in fetus and newborn infants Hb F binds O2 at lower tension than Hb A
→ Hb F has a higher affinity to O2
After birth, Hb F is replaced by Hb A during the first few months of
life.

 Hb S – in β-globin chain Glu is replaced by Val

= an abnormal Hb typical for sickle cell anemia

Figure is found at http://en.wikipedia.org/wiki/Sickle-cell_disease

 Fetal Hb vs. adult Hb

Pressure units:
1 mmHg = 1 Torr
1 mmHg = 133.22 Pa
1 Pa = 0.0075 mmHg

Figure is found at http://en.wikipedia.org/wiki/Fetal_hemoglobin

 Derivatives of hemoglobin
 Oxyhemoglobin (oxyHb) = Hb with O2

 Deoxyhemoglobin (deoxyHb) = Hb without O2

 Methemoglobin (metHb) contains Fe3+ instead of Fe2+ in heme groups

 Carbonylhemoglobin (HbCO) – CO binds to Fe2+ in heme in case of

CO poisoning or smoking. CO has 200x higher affinity to Fe2+ than O2.

 Carbaminohemoglobin (HbCO2) - CO2 is non-covalently bound to globin

chain of Hb. HbCO2 transports CO2 in blood (about 23%).

 Glycohemoglobin (HbA1c) is formed spontaneously by nonenzymatic

reaction with Glc. People with DM have more HbA1c than normal (› 7%).
Measurement of blood HbA1c is useful to get info about long-term
control of glycemia.
 Function of hemoglobin

• Hb is a buffer (Hb/Hb-H+) in the erythrocytes

• Hb is a carrier of O2 and CO2
Binding of O2 is a cooperative. Hb binds O2 weakly at low oxygen
pressures and tightly at high pressures. The binding of the first O2
to Hb enhances the binding futher O2 molecules → allosteric
effect → S-shaped (sigmoidal) saturation curve of Hb

Figures are found at http://www.chemistry.wustl.edu/~edudev/labTutorials/Hemoglobin/

MetalComplexinBlood.html and http://en.wikipedia.org/wiki/Hemoglobin
 Process of O2 binding to Hb
Hb can exist in 2 different forms: T-form and R-form.
T-form (T = „tense“) has a much lower oxygen affinity than the R-form.
The subunits of Hb are held together by electrostatic interactions. The
binding of the first O2 molecule to subunit of the T-form leads to a local
conformational change that weakens the association between the subunits
→ R-form („relaxed“) of Hb.

Increasing of oxygen partial pressure causes the conversion of T-form

to R-form.
T  R
Hb + ↑pO2  HbO2
 Agents that influence oxygen binding
● 2,3-bisphosphoglycerate (2,3-BPG) only binds to deoxyHb
(β-chains) → deoxyHb is thus stabilized
● H+ ions (lower pH) – binding of H+ by Hb lowers its affinity for
O2 → Bohr effect
● CO2 – high CO2 levels in the plasma also result in a right shift of
saturation curve = Bohr effect

Figures are found at http://en.wikipedia.org/wiki/Bohr_Effect and

http://themedicalbiochemistrypage.org/hemoglobin-myoglobin.html
 Transport of O2 and CO2 in lungs

Figure is found at http://www.mfi.ku.dk/ppaulev/chapter15/Chapter15.html

 Transport of O2 and CO2 in tissues

Figure is found at http://www.mfi.ku.dk/ppaulev/chapter15/Chapter15.htm

 Metabolism of erythrocyte
• ATP is generated by anaerobic glycolysis → ATP is used for ion
transport across the cell membrane
• glycolysis produces 2,3-BPG and lactate
• approx. 5 to 10% of Glc is metabolized by hexose monophosphate
pathway → production of NADPH → it is used to maintain
glutathione in the reduced state

Figure is found at http://themedicalbiochemistrypage.org/hemoglobin-myoglobin.html

 Porphyrias - disturbances of heme synthesis
• are hereditary or acquired disturbances of heme
synthesis

• in all cases there is an identifiable abnormality of

the enzymes which synthesize heme

• this leads to accumulation of intermediates of the

pathway and a deficiency of heme → excretion of
heme precursors in feces or urine, giving them a dark
red color

● accumulation of porphyrinogens in the skin can lead to

photosensitivity

• the neurological symptoms

 Thalassemias – inherited autosomal
recessive blood diaseases
• genetic defect results in reduced rate of synthesis
of α- or β-globin chain → it causes the formation of
abnormal Hb molecules → anemia
• are prevalent in populations where malaria was
endemic – Arab-Americans, people of Mediterranean
origin and Asians
• genetic counseling and genetic testing is
recommended for families that carry a thalassemia
trait