BIOSYNTHESIS & BIOSYNTHESIS & CATABOLISM OF CATABOLISM OF HEMOGLOBIN HEMOGLOBIN

Abdul Salam M. SofroAbdul Salam M. Sofro

Faculty of MedicineFaculty of Medicine

YARSI University JakartaYARSI University Jakarta

Learning objectivesLearning objectives

By the end of learning, students are By the end of learning, students are expected to understand:expected to understand: Molecular structure and function of Molecular structure and function of

hemoglobinhemoglobin Biosynthesis of hemoglobinBiosynthesis of hemoglobin Catabolic process and the fate of Catabolic process and the fate of

hemoglobin catabolic products hemoglobin catabolic products

Hemoglobin in blood

Blood cells development

GeneralGeneral

Hemoglobin (four subunits) and its Hemoglobin (four subunits) and its similar molecule myoglobin (one similar molecule myoglobin (one subunit) are iron-containing heme subunit) are iron-containing heme proteins proteins consists of apoprotein & consists of apoprotein & non-protein hemenon-protein heme

These heme proteins function in These heme proteins function in oxygen binding, oxygen transport, oxygen binding, oxygen transport, electron transport & photosynthesis electron transport & photosynthesis carried out by heme (a cyclic carried out by heme (a cyclic tetrapyrrole) & its ferrous iron (at the tetrapyrrole) & its ferrous iron (at the center of the planar ring) center of the planar ring)

Hemoglobin structure

Hemoglobin function

The Molecular structure is The Molecular structure is similar to Myoglobinsimilar to Myoglobin : :

MW 17,000 ; a monomer of protein MW 17,000 ; a monomer of protein with 153 AA residueswith 153 AA residues

stores oxygen in red muscle tissue stores oxygen in red muscle tissue will be released under condition of will be released under condition of oxygen deprivation (eg. Severe oxygen deprivation (eg. Severe aexercise) and used by muscle aexercise) and used by muscle mitochondria for ATP synthesismitochondria for ATP synthesis

75% of the AA residues are 75% of the AA residues are present in 8 present in 8 -helix (helix A to H)-helix (helix A to H)

Histidin F8 and E7 perform unique Histidin F8 and E7 perform unique roles in oxygen bindingroles in oxygen binding

Oxygen-binding curve for Oxygen-binding curve for myoglobin is hyperbolic myoglobin is hyperbolic

Hemoglobin:Hemoglobin:

Transports oxygen, COTransports oxygen, CO22 & protons & protons Its allosteric properties results from its Its allosteric properties results from its

quaternary structuresquaternary structures A tetramer composed of pairs of A tetramer composed of pairs of

different polypeptides/subunits (different polypeptides/subunits (, , , , , , etc. globin chains) etc. globin chains) a pair of globin a pair of globin chain product of gene cluster in chain product of gene cluster in chromosome 11 & a pair of globin chromosome 11 & a pair of globin chain product of gene cluster in chain product of gene cluster in chromosome 16 chromosome 16

Hb binds 2 protons for every 4 oxygen Hb binds 2 protons for every 4 oxygen molecules released & thus contributes molecules released & thus contributes significantly to buffering capacity of blood significantly to buffering capacity of blood increase in proton concentration increase in proton concentration promotes oxygen release, while increase promotes oxygen release, while increase in Pin PO2O2 promotes proton release. promotes proton release.

At the lungs, oxygenation of Hb is At the lungs, oxygenation of Hb is accompanied by expulsion and accompanied by expulsion and subsequent expiration of COsubsequent expiration of CO2 2 Bohrs Bohrs

effect (a reversible phenomenon with that effect (a reversible phenomenon with that in the peripheral tissues)in the peripheral tissues)

2,3-Bisphosphoglycerate (BPG) in Hb2,3-Bisphosphoglycerate (BPG) in Hb Formed from glycolytic intermediate Formed from glycolytic intermediate

1,3-bisphosphoglycerate1,3-bisphosphoglycerate One molecule of BPG is bound per Hb One molecule of BPG is bound per Hb

tetramer in the central cavity tetramer in the central cavity the the space is wide enough when Hb is in the space is wide enough when Hb is in the T form (deoxygenated)T form (deoxygenated)

Binds more weakly to fetal Hb than to Binds more weakly to fetal Hb than to adult Hb adult Hb

Increase concentration of BPG lowers Increase concentration of BPG lowers the affinity of Hb for oxygen the affinity of Hb for oxygen (decreases P(decreases P5050) ) increasing the increasing the ability of Hb to release oxygen at the ability of Hb to release oxygen at the tissuestissues

As COAs CO2 2 is absorbed in the blood, the is absorbed in the blood, the

carbonic anhydrase (CA) in erythrocyte carbonic anhydrase (CA) in erythrocyte catalyzes the formation of carbonic acid, catalyzes the formation of carbonic acid, which in turn rapidly dissociate into which in turn rapidly dissociate into bicarbonate and a proton. To avoid bicarbonate and a proton. To avoid increasing the acidity of blood, a buffering increasing the acidity of blood, a buffering system must absorb these excess protons system must absorb these excess protons this is carried out by Hb this is carried out by Hb

COCO22 + H + H22O O H H22COCO33 HCOHCO33-- + H + H++

CA spontaneous

Mutant human HbMutant human Hb Causes hemoglobinopathy (when Causes hemoglobinopathy (when

biologic function is altered)biologic function is altered) Due to mutations in the gene that Due to mutations in the gene that

code for globin chains:code for globin chains:Structurally abnormal Hb (HbM, Structurally abnormal Hb (HbM, HbS, HbE, HbC etc)HbS, HbE, HbC etc)

Reduced synthesis of Hb Reduced synthesis of Hb (thalassemias) (thalassemias)

Diagnosed by special method (e.g. Diagnosed by special method (e.g. molecular diagnosis)molecular diagnosis)

Batak

Melayu

Minang

Palembang

Bangka

Dayak

Banjar

Palu

Minahasa

Jawa

TenggerSumbawaBali

SumbaSasak

Alor

Toraja

Gambar . Pola distribusi dan prevalensi trait thalassemia- dan hemoglobin-E pada berbagai populasi di Indonesia. * adalah hemoglobin OIna.

1,5 0

3,7

5,2

2,9

4,3

9,2 6,5

5,4 4,5

3,2 4,8

0 10,6

3,1 1,5

0 0

0 1,7

1,2 3,7

0 4*

1,2 6,1

2,9 4,32,5 36,6

5,1 6,8 0 0

= trait thalassemia-

= trait hemoglobin-E

HemeHeme

In addition to the heme b found in hemoglobin, there are three different forms of heme found in

cytochromes such as those involved in the process of oxidative phosphorylation.

Cytochromes of the c type contain a modified iron protoporphyrin IX known as heme c. In heme c

the 2 vinyl (C=C) side chains are covalently bonded to cysteine sulfhydryl residues of the apoprotein. Only cytochromes of the c type

contain covalently bound heme. Heme a is also a modified iron protoporphyrin IX. Heme a is found

in cytochromes of the a type and in the chlorophyll of green plants

Biosynthesis of hemeBiosynthesis of heme

Protoporphyrin IXProtoporphyrin IX

The sythesis of heme is a complex process that involves multiple enzymatic steps. The process begins in the mitochondrion with the condensation of succinyl-CoA and glycine to form 5-aminolevulinic acid. A series of steps in the cytoplasm produce coproporphrynogen III, which re-enters the mitochondrion. The final enzymatic steps produce heme.

Synthesis of Porphobilinogen and Heme Synthesis of Porphobilinogen and Heme

http://themedicalbiochemistrypage.org/heme-porphyrin.html

GlobinGlobin

a polypeptide chain (protein)a polypeptide chain (protein) Various types of polypeptide chain:Various types of polypeptide chain:

Alpha globinAlpha globin Beta globinBeta globin Gamma globinGamma globin Delta globinDelta globin Epsilon globinEpsilon globin Zetta globinZetta globin Teta globinTeta globin

Globin genesGlobin genes

Chromosome 11 Chromosome 11

((- cluster): - cluster):

--GG--AA- - ---- Chromosome 16 Chromosome 16

((-cluster):-cluster):

22--11--22--11--22--11--

2

2 22 22

2

2

1 1 1G lo b in G e ne s :

Hb typ e s :

Em b ryo

(G o we r-I) (Po rtla nd ) (G o we r-II)

C ha ins Synthe size d

5 ' 3 '

Chromosome #16

2 2 222 2 2 2

Fe tus Ad ult

(Hb -F) (Hb -A ) (Hb -A)2

3 '5 ' G

G

G

A

A

A

G lo b in G e ne s :

Hb typ e s :

C ha ins Synthe size d

Chromosome #11

50

30

10

6 18 30 6 18 30 42prenata l age (wks)

% of to ta lg lobinsynthesis

birth

postnata l age (w ks)

Types of hemoglobinTypes of hemoglobin

Hb Gower 1Hb Gower 1 = = 2222 Hb PortlandHb Portland = = 2222 Hb Gower 2Hb Gower 2 = = 2222 Hb Fetal (HbF)Hb Fetal (HbF) = = 2222 Hb Adult (HbA)Hb Adult (HbA) = = 2222 Hb Adult minor (HbA2)Hb Adult minor (HbA2) = = 2222

Catabolism of Heme Catabolism of Heme

Heme breakdownHeme breakdown

During its 120 day life span the erythrocyte During its 120 day life span the erythrocyte has traveled 200-300 miles. The process of has traveled 200-300 miles. The process of aging is called senescence. aging is called senescence.

Enzyme activity decreases (esp. glycolytic Enzyme activity decreases (esp. glycolytic enzyme which helps break down glucose, enzyme which helps break down glucose, the source of erythrocyte energy), and the the source of erythrocyte energy), and the cell looses its deformability. cell looses its deformability.

MCHC (mean corpuscular hemoglobin MCHC (mean corpuscular hemoglobin concentration) increases, the cell becomes concentration) increases, the cell becomes rounder, and the MCV mean corpuscular rounder, and the MCV mean corpuscular volume) decreases. volume) decreases.

90% of destruction of senescent 90% of destruction of senescent Erythrocytes occurs by extravascular Erythrocytes occurs by extravascular hemolysis. Macrophages of the hemolysis. Macrophages of the mononuclear phagocyte system remove mononuclear phagocyte system remove them from circulation. them from circulation.

Macrophages of the spleen are Macrophages of the spleen are especially active in removing aging, dead especially active in removing aging, dead and abnormal erythrocytes (e.g. cells and abnormal erythrocytes (e.g. cells containing Heinz bodies or Howell-Jolly containing Heinz bodies or Howell-Jolly bodies, siderocytes, target cells, bodies, siderocytes, target cells, schistocytes, tear drop cells and schistocytes, tear drop cells and antibody-coated erythrocytes).antibody-coated erythrocytes).

Normally, senescent red blood cells and Normally, senescent red blood cells and heme from other sources are engulfed by heme from other sources are engulfed by cells of the reticuloendothelial system. The cells of the reticuloendothelial system. The globin is recycled or converted into amino globin is recycled or converted into amino acids, which in turn are recycled or acids, which in turn are recycled or catabolized as required. catabolized as required.

Heme is oxidized, with the heme ring being Heme is oxidized, with the heme ring being opened by the endoplasmic reticulum opened by the endoplasmic reticulum enzyme, heme oxygenase. The oxidation enzyme, heme oxygenase. The oxidation step requires heme as a substrate, and any step requires heme as a substrate, and any hemin (Fe3+) is reduced to heme (Fe2+) hemin (Fe3+) is reduced to heme (Fe2+) prior to oxidation by heme oxygenaseprior to oxidation by heme oxygenase

Pathway for the degradation of heme to bilirubin.

Substituents:

M = methyl, P = proprionic, V = vinyl

In individuals with abnormally high red In individuals with abnormally high red cell lysis, or liver damage with obstruction cell lysis, or liver damage with obstruction of the bile duct, the bilirubin and its of the bile duct, the bilirubin and its precursors accumulate in the circulation; precursors accumulate in the circulation; the result is the result is hyperbilirubinemiahyperbilirubinemia, the , the cause of the abnormal yellowish cause of the abnormal yellowish pigmentation of the eyes and tissues pigmentation of the eyes and tissues known as known as jaundicejaundice. .

The protoporphyrin ring of heme is The protoporphyrin ring of heme is disassembled and from the body. Its disassembled and from the body. Its alpha carbon is exhaled in the form of alpha carbon is exhaled in the form of COCO22. The opened tetrapyrrole, biliverdin, . The opened tetrapyrrole, biliverdin,

is converted to bilirubin which is then is converted to bilirubin which is then carried to the liver by the plasma protein, carried to the liver by the plasma protein, albumin. albumin.

In the liver bilirubin is conjugated to In the liver bilirubin is conjugated to glucuronide to make it water soluble and glucuronide to make it water soluble and excreted along with bile into the intestines. excreted along with bile into the intestines. In the intestines it is converted by bacteria In the intestines it is converted by bacteria into stercobilinogen and excreted in the into stercobilinogen and excreted in the stool; some is eliminated as urobilinogen in stool; some is eliminated as urobilinogen in the urine. the urine.

Stercobilinogen and urobilinogen give feces Stercobilinogen and urobilinogen give feces and urine their color.and urine their color.

UnconjugatedUnconjugated bilirubin (prehepatic) and bilirubin (prehepatic) and conjugated conjugated bilirubin (posthepatic) are bilirubin (posthepatic) are measured in serum as indirect measured in serum as indirect (unconjugated) and direct (conjugated) (unconjugated) and direct (conjugated) bilirubin; used to monitor amount of bilirubin; used to monitor amount of hemolysis.hemolysis.

Bilirubin and its catabolic products are Bilirubin and its catabolic products are collectively known as the collectively known as the bile pigments. bile pigments.

Intravascular hemolysisIntravascular hemolysis

About 10% of normal erythrocyte About 10% of normal erythrocyte destruction occurs by intravascular destruction occurs by intravascular hemolysis.  hemolysis. 

In circulation the red cell is subjected to In circulation the red cell is subjected to metabolic and mechanical stresses: metabolic and mechanical stresses: turbulence, endothelial damage and fibrin turbulence, endothelial damage and fibrin deposition, incompatibility due to deposition, incompatibility due to transfusion errors resulting in red cell transfusion errors resulting in red cell fragmentation (schistocytes) and/or fragmentation (schistocytes) and/or intravascular hemolysis.intravascular hemolysis.

When the erythrocyte ruptures, When the erythrocyte ruptures, hemoglobin is released into the blood. The hemoglobin is released into the blood. The hemoglobin dissociates into alpha-beta hemoglobin dissociates into alpha-beta dimers and is picked up haptoglobin, a dimers and is picked up haptoglobin, a protein carrier, to prevent renal excretion of protein carrier, to prevent renal excretion of hemoglobin. hemoglobin.

Haptoglobin carries the hemoglobin to the Haptoglobin carries the hemoglobin to the liver for further catabolism where the liver for further catabolism where the process proceeds as with extravascular process proceeds as with extravascular hemolysis.hemolysis.

As haptoglobin is depleted, unbound As haptoglobin is depleted, unbound hemoglobin dimers appear in the plasma hemoglobin dimers appear in the plasma (hemoglobinemia) and are reabsorbed by (hemoglobinemia) and are reabsorbed by the kidney up to a certain level and the kidney up to a certain level and converted to hemosiderin; beyond this converted to hemosiderin; beyond this level hemoglobin shows up in the urine level hemoglobin shows up in the urine (hemoglobinuria)(hemoglobinuria)

Intravascular hemolysis results in pink, red Intravascular hemolysis results in pink, red or brown plasma (hemoglobinemia). Urine or brown plasma (hemoglobinemia). Urine may also show red color (hemoglobinuria).may also show red color (hemoglobinuria).

http://diaglab.vet.cornell.edu/clinpath/modules/chem/images/bilirubin%20metabolism.jpg

Clinical Aspect of Heme MetabolismClinical Aspect of Heme Metabolism

Clinical problems associated with heme Clinical problems associated with heme metabolism are of two types. metabolism are of two types. Disorders that arise from defects in the Disorders that arise from defects in the

enzymes of heme biosynthesis are termed enzymes of heme biosynthesis are termed the the porphyriasporphyrias and cause elevations in the and cause elevations in the serum and urine content of intermediates in serum and urine content of intermediates in heme synthesis. heme synthesis.

Inherited disorders in bilirubin metabolism Inherited disorders in bilirubin metabolism lead to lead to hyperbilirubinemiahyperbilirubinemia

PorphyriaPorphyria Enzyme DefectEnzyme Defect Primary SymptomPrimary Symptom

Erythroid ClassErythroid Class

X-linked sideroblastic X-linked sideroblastic anemia, XLSAanemia, XLSA

δ-aminolevulinic acid δ-aminolevulinic acid synthase 2, ALAS2synthase 2, ALAS2

progressive iron progressive iron accumulation, fatal if not accumulation, fatal if not treatedtreated

Congenital Congenital erythropoietic erythropoietic porphyria, CEPporphyria, CEP

uroporphyrinogen III uroporphyrinogen III cosynthasecosynthase photosensitivityphotosensitivity

Erythropoietic Erythropoietic protoporphyria, EPPprotoporphyria, EPP ferrochelataseferrochelatase photosensitivityphotosensitivity

Hepatic ClassHepatic Class

ALA dehydratase deficient porphyria, ALA dehydratase deficient porphyria, ADPADP

ALA dehydratase: also called ALA dehydratase: also called porphobilinogen synthaseporphobilinogen synthase neurovisceralneurovisceral

Acute intermittent porphyria, AIPAcute intermittent porphyria, AIP

PBG deaminase: also called PBG deaminase: also called hydroxymethylbilane hydroxymethylbilane synthase or rarely synthase or rarely uroporphyrinogen I synthaseuroporphyrinogen I synthase

neurovisceralneurovisceral

Hereditary coproporphyria, HCPHereditary coproporphyria, HCP coproporphyrinogen oxidasecoproporphyrinogen oxidaseneurovisceral, neurovisceral, some some photosensitivityphotosensitivity

Variegate porphyria, VPVariegate porphyria, VP protoporphyrinogen oxidaseprotoporphyrinogen oxidaseneurovisceral, neurovisceral, some some photosensitivityphotosensitivity

Porphyria cutanea tarda type I, PCT Porphyria cutanea tarda type I, PCT type I, also called the sporadic type type I, also called the sporadic type PCTPCT

hepatic uroporphyrinogen hepatic uroporphyrinogen decarboxylasedecarboxylase photosensitivityphotosensitivity

Porphyria cutanea tarda type II, PCT Porphyria cutanea tarda type II, PCT type II, also called the familial type type II, also called the familial type PCT, may also be referred to as PCT, may also be referred to as hepatoerythropoietic porphyria, HEPhepatoerythropoietic porphyria, HEP

uroporphyrinogen uroporphyrinogen decarboxylase in non-hepatic decarboxylase in non-hepatic tissuestissues

photosensitivityphotosensitivity, some , some neurovisceralneurovisceral