9/6/2022

HEMOGLOBIN (HB)

 is a hemoprotein only found in the cytoplasm of

erythrocytes (ery)
HEMOGLOBIN, IRON  transports O2 and CO2 between lungs and
various tissues
AND PORPHYRIAS  normal concentration of Hb in the blood:
adult males 135 – 175 g/L
adult females 120 – 168 g/L

STRUCTURE OF HEMOGLOBIN HEME STRUCTURE

HEME IS A METALOPORPHYRINE
(CYCLIC TETRAPYRROLE)
• Hb is a spherical molecule consisting of 4 peptide
subunits (globins) = quartenary structure Heme contains:
• Hb of adults (Hb A) is a tetramer consisting of 2 -  conjugated system of
and 2 β-globins → each globin contains 1 heme group double bonds → red
with a central Fe2+ ion (ferrous ion) colour
 4 nitrogen (N) atoms

 1 iron cation (Fe2+)

→ bound in the middle of
tetrapyrrole skelet by methine bridge
coordination covalent pyrrole ring
bonds

PROPERTIES OF IRON IN IN WHICH COMPOUNDS CAN WE FIND

HEME A HEME GROUP?

• Coordination number of iron  Hemoproteins

in heme = 6 • Hemoglobin (Hb)
• Myoglobin (Mb)
6 bonds:
• Cytochromes
• 4x pyrrole ring (A,B,C,D)
• 1x link to a protein • Catalases (decomposition of 2
H2O2 to 2 H2O and O2)
• 1x link to an oxygen
• Peroxidases

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Δ-AMINOLEVULINIC ACID
MYOGLOBIN (MB) (ALA)
• synthesis of heme starts in mitochondria
• succinyl-CoA and Gly undergo a condensation → ALA
• reaction is catalyzed by enzyme ALA synthase
• 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

PORPHOBILINOGEN (PBG) UROPORPHYRINOGEN →

COPROPORPHYRINOGEN III
• ALA leaves the mitochondria → cytoplasm
• 2x ALA condense together to form porphobilinogen
• reaction is catalyzed by porphobilinogen synthase • enzyme hydroxymethylbilane synthase catalyzes
(ALA dehydratase) 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

PROTOPORPHYRINOGEN IX → PROTOPORPHYRIN IX
FINAL FORMATION OF HEME
• oxidation of protoporphyrinogen IX produces the
conjugated π-electrone system of protoporphyrin IX
• Fe2+ is incorporated into protoporphyrin IX
• reaction is catalyzed by enzyme ferrochelatase

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REGULATION OF HEME BIOSYNTHESIS HEME BIOSYNTHESIS - REPETITION

• in bone marrow (85% of Hb) and liver (cytochromes)

 ALA synthase is a key regulatory enzyme
• cell location: mitochondria / cytoplasm / mitochondria
● it is an allosteric enzyme that is inhibited by an end product - heme
(feedback inhibition) • substrates: succinyl-CoA + glycine
● requires pyridoxal phosphate as a coenzyme • important intermediates:
● certain drugs and steroid hormones can increase heme synthesis  -aminolevulinic acid (= 5-aminolevulinic acid, ALA)
Porphobilinogen synthase is inhibited by lead ions Pb2+ in case of lead  porphobilinogen (PBG = pyrrole derivate)
poisoning.
 uroporphyrinogen III (= porphyrinogen – heme precursor)
 Ferrochelatase (heme synthase) can be also inhibited by Pb
2+. Its

activity is influenced by availability of Fe2+ and ascorbic acid.  protoporphyrin IX (= direct heme precursor)
● key regulatory enzyme: ALA synthase

HEMOGLOBIN DEGRADATION CONVERTION OF HEME TO BILIRUBIN

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.
green
Hb is degraded to:
● globins → AAs → metabolism
● heme → bilirubin
● Fe2+ → transported with transferrin and used in the next heme red-orange
biosynthesis

Not only Hb but other hemoproteins also contain heme groups

which are degraded by the same pathway.

FUTHER FATE OF BILIRUBIN IN INTESTINE:

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  Urobilinogen and stercobilinogen can be:
albumin („nonconjugated Bil“).
a) reabsorbed and returned to the liver
↓
 (= enterohepatic circulation)

LIVER
In the hepatocytes, Bil is conjugated by 2 molecules of glucuronic acid  b) oxidized (in the presence of O2) to pigments urobilin
→ bilirubin diglucuronide (soluble in water, „conjugated Bil“) (orange) and stercobilin (yellow) → they are excreted in the
↓ stool
BILE
↓  Urobilinogen also appears in the urine.
INTESTINE
Bil is reduced to urobilinogen and stercobilinogen

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CLINICAL CORRELATIONS
DETERMINATION OF
bilirubin-diglucuronide = conjugated bilirubin BILIRUBIN (BIL) IN SERUM
is soluble in water → „direct bilirubin“ Blood tests
 Bil reacts directly when reagents are added to the blood sample →
conjugated bilirubin = direct Bil (up to 3.4 µmol/L)

 Bile pigments:
 free Bil does not react to the reagents until alcohol (methanol) or
• bilirubin caffeine is added to the solution. Therefore, the measurement of this
• urobilin type of bilirubin is indirect → unconjugated bilirubin = indirect Bil (up
to 13.6 µmol/L)
• stercobilin

 total bilirubin measures both unconjugated and conjugated Bil

(normal value up to 17 µmol/L).

TYPES OF HEMOGLOBIN FETAL HB VS. ADULT HB

 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.
Pressure units:
HbA2 (2 α, 2 δ) is a minor form of Hb in adults. It forms only 2 – 3% of 1 mmHg = 1 Torr
1 mmHg = 133.22 Pa
a total Hb A. 1 Pa = 0.0075 mmHg

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

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

= an abnormal Hb typical for sickle cell anemia

DERIVATIVES OF HEMOGLOBIN
 Oxyhemoglobin (oxyHb) = Hb with O2 FUNCTION OF HEMOGLOBIN
 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%). • Hb is a buffer (Hb/Hb-H+) in the erythrocytes
• Hb is a carrier of O2 and CO2
 Glycohemoglobin (HbA1c) is formed spontaneously by nonenzymatic Binding of O2 is a cooperative. Hb binds O2 weakly at low oxygen
reaction with Glc. People with DM have more HbA1c than normal (› 7%). pressures and tightly at high pressures. The binding of the first O2 to
Measurement of blood HbA1c is useful to get info about long-term Hb enhances the binding futher O2 molecules → allosteric effect →
control of glycemia. S-shaped (sigmoidal) saturation curve of Hb

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AGENTS THAT INFLUENCE OXYGEN BINDING

PROCESS OF O2 BINDING TO HB
 ● 2,3-bisphosphoglycerate (2,3-BPG) only binds to deoxyHb
Hb can exist in 2 different forms: T-form and R-form.  (β-chains) → deoxyHb is thus stabilized
T-form (T = „tense“) has a much lower oxygen affinity than the R-  ● H+ ions (lower pH) – binding of H+ by Hb lowers its affinity for O2
form. The subunits of Hb are held together by electrostatic → Bohr effect
interactions. The binding of the first O2 molecule to subunit of the T-
 ● CO2 – high CO2 levels in the plasma also result in a right shift of
form leads to a local conformational change that weakens the saturation curve = Bohr effect
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

TRANSPORT OF O2 AND CO2 IN LUNGS

METABOLISM OF ERYTHROCYTE
 Porphyrins are cyclic compounds that bind metal ions (usually
Fe2+ or Fe3+)
• ATP is generated by anaerobic glycolysis → ATP is used for ion transport  Porphyrin + Metal = Metalloporphyrin
across the cell membrane
 Most prevalent metalloporphyrin in humans is heme (metal here
• glycolysis produces 2,3-BPG and lactate is iron ion)
• approx. 5 to 10% of Glc is metabolized by hexose monophosphate pathway
→ production of NADPH → it is used to maintain glutathione in the
Heme consists of:
reduced state  One ferrous ion (Fe2+) in the centre
 Protoporphyrin IX (a tetrapyrrole ring)
 The role of heme is dictated by the environment created by the
three dimensional structure of protein
Heme is the prosthetic group of hemoglobin, myoglobin,
cytochromes, catalase, tryptophan pyrrolase
* So, heme + globin protein = hemoglobin*

WHAT ARE PORPHYRINS ?

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STRUCTURE OF PORPHYRINS
 Porphyrins are cyclic molecules formed by 4 pyrrole (tetrapyrrole) rings linked by methenyl
bridges.

 Different porphyrins vary in the nature of side chains that are attached to each of the 4
pyrrole rings
Protoporphyrin IX contains vinyl, methyl & propionate

Distribution of side chains

- Side chains can be ordered around tetrapyrrole nucleus in 4 different ways designated
Pyrrol
I, II, III & IV series. Side Methenyl e
Only type III porphyrins are physiologically important in humans
Chains bridge ring Structure of Porphyrin
- Protoporphyrin IX is a member of type III series

 Porphyrinogens
are porphyrin precursors
intermediate between porphobilinogen & protoporphyrin

Porphobilinogen Porphyrinogens Protoporphyrins

Determine the type of porphyrin

Overview of Heme Synthesis

Structure and Properties of Iron Protoporphyrin IX

propionate

methyl

pyrrole ring

vinyl

Derived from protoporphyrin IX

Pattern of side chains defines isomer
Binds metals: Heme- Fe2+ (ferrous)
Hemin- Fe3+ (ferric) Heme synthesis occurs in all cells due to the requirement for heme as a
Zinc protoporphyrin (ZnPP)- Zn2+ prosthetic group on enzymes and electron transport chain. By weight, the
major locations of heme synthesis are the liver and the erythroid progenitor
Extended conjugation across ring system cells of the bone marrow.

PORPHYRIAS
PORPHYRIAS - DISTURBANCES OF HEME SYNTHESIS

Porphyria are rare inherited defects in heme synthesis.

• are hereditary or acquired disturbances of heme synthesis
An inherited defect in an enzyme of heme synthesis results in accumulation of
• in all cases there is an identifiable abnormality of the enzymes one
which synthesize heme or more of porphyrin precursors depending on location of block of the heme
synthesis pathway.
• 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 These precursors increase in blood & appear in urine of patients.

● accumulation of porphyrinogens in the skin can lead to Porphyria means purple colour caused by pigment-like porphyrins in urine of
photosensitivity patients. (Diagnosed by lab investigation)

• the neurological symptoms Most porphyrias show a prevalent autosomal dominant pattern, except
congenital eythropoietic porphyria, which is recessive

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Clinical manifestations of porphyrias:

 Chronic hepatic porphyria
Two types of porphyrias: erythropoietic (bone marrow) & hepatic  The most common type of porphyria
Hepatic porphyrias are: acute & chronic porrphyrias
 a deficiency in uroporphyrinogen decarboxylase
Generally, individuals with an enzyme defect prior to the synthesis of the
tetrapyrroles manifest abdominal and neuropsychiatric signs  Clinical expression of the enzyme deficiency is influenced by various
factors, such as exposure to sunlight, the presence of hepatitis B or C
Those with tetrapyrrole intermediates show photosensitivity with
formation  Clinical onset is during the fourth or fifth decade of life.
of reactive oxygen species (ROS) that damage membranes by oxidation
resulting  Porphyrin accumulation leads to cutaneous symptoms and urine that is
in the following effects: red to brown in natural light and pink to red in fluorescent light
- Skin blisters, itches (pruritis)
- Skin may darken, grow hair (hypertrichosis)
PORPHYRIA CUTANEA TARDA

THALASSEMIAS – INHERITED
AUTOSOMAL RECESSIVE BLOOD
DIASEASES
e.g. Acute Intermittent Porphyria

 Porphyrias leading to accumulation of ALA and porphobilinogen cause • genetic defect results in reduced rate of synthesis of α- or β-
abdominal pain and neuropsychiatric disturbances, ranging from globin chain → it causes the formation of abnormal Hb molecules →
anxiety to delirium. anemia
• are prevalent in populations where malaria was endemic – Arab-
Americans, people of Mediterranean origin and Asians
 Symptoms of the acute hepatic porphyrias are often precipitated by
administration of drugs such as barbiturates and ethanol. • genetic counseling and genetic testing is recommended for families
that carry a thalassemia trait

ACUTE HEPATIC PORPHYRIAS