1. Structure, function and metabolism of hemoglobin
Pavla Balínová
Figure was adopted from

2. Hemoglobin (Hb)
is a hemoprotein only found in the cytoplasm of erythrocytes (ery)
transports O2 and CO2 between lungs and various 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

3. 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

4. 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
Overheads/Fig%2015-A.jpg

5. 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

6. 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

7. 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

8. 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

9. 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

10. 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

11. 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.

12. 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.

13. Final formation of heme
Fe2+ is incorporated into protoporphyrin IX
reaction is catalyzed by enzyme ferrochelatase
Figure was assumed from
ferrochelatase.html

14. 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.

15. Figure was assumed from book Color Atlas of Biochemistry / J
Figure was assumed from book Color Atlas of Biochemistry / J. Koolman, K.H.Röhm. Thieme 2005.
ISBN

16. 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.

17. Convertion of heme to bilirubin
green
red-orange
Figure is found at

18. 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

19. bilirubin-diglucuronide = conjugated bilirubin is soluble in water → „direct bilirubin“
Figure is found at

20. In intestine: Urobilinogen and stercobilinogen can be:
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.

21. Bile pigments: bilirubin urobilin stercobilin
Figure was assumed from book Color Atlas of Biochemistry / J. Koolman, K.H.Röhm. Thieme 2005.

22. 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).

23. 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

24. Fetal Hb vs. adult Hb Pressure units: 1 mmHg = 1 Torr
1 mmHg = Pa
1 Pa = mmHg
Figure is found at

25. 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.

26. 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
MetalComplexinBlood.html and

27. 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

28. 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 and

29. Transport of O2 and CO2 in lungs
Figure is found at

30. Transport of O2 and CO2 in tissues
Figure is found at

31. 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

32. 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

33. 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