Hemoglobin synthesis and function

MLS 307 Introduction to Clinical Hematology

Graphic found at http://www.kacr.or.kr/img/gene_expression/hemoglobin.jpg, 2001.

Hemoglobin synthesis
The hemoglobins are red globular proteins, which have a molecular weight of about 68,000 and comprise almost one third of the weight of a red cell. The hemoglobin is composed of heme and globin.

Hemoglobin synthesis
Main fxn:
Carry O2 Carry CO2 Each red cell contains approximately 640 million Hb molecules.

Hemoglobin synthesis
65% -erythroblasts 35% -reticulocyte Heme synthesis occurs largely in the mitochondria. Globin synthesis occurs in the polyribosomes. Although heme and globin synthesis occur separately within developing red cell precursors, their rates of synthesis are carefully coordinated to ensure optimal efficiency of Hb assembly.

Hemoglobin Structure
Conjugated protein
Dimeric globin
2 a-globin chains 2 b-globin chains

4 heme groups
Four iron (ferrous) molecules Four protoporphyrin IX rings

1 2,3-DPG

Graphic accessed http://porpax.bio.miami.edu/~cmallery/150/chemistry/hemoglobin.jpg, 2008.

Factoid: 95% of a red cells dry weight is hemoglobin!

Hemoglobin Structure
Conformation
Primary = type, number, and sequence of amino acids Secondary = chains arrangements Tertiary = 3-D arrangement of 2 structure Quaternary = complete structure
Graphic accessed http://matcmadison.edu/biotech/resources/proteins/labManual/images/220_04_114.png, 2004

Hemoglobin Synthesis: Globin

Chains of amino acids Each protein is characterized by the number and sequence of amino acids Each globin chain is designated by a greek letter: a, b, g, d, e, z Genetics Chromosome 16 = a, z Chromosome 11 = b, d, e, g

Synthesis in cytosol

Graphic accessed http://www.med.virginia.edu/medicine/clinical/pathology/educ/innes/text/nh/globin.html, 2001.

Globin gene clusters

Synthesis of globin
Embryonic Hemoglobin Gower I ( z2e2) Hemoglobin Portland ( z2g2) Hemoglobin Gower II (a2e2) Fetal : HbF (a2g2), HbA (a2b2) Adult : HbA, HbA2 ( a2d2), HbF.

Globin chain switch

Alpha & beta chains

Adult hemoblobin
Hb A structure a2b2 Hb A2 a2d2 Hb F a2g2

Normal %

96-98 %

1.5-3.2 %

0.5-0.8 %

Quantitative disorders of globin synthesis are called Thalassemia

Qualitative disorders are called Hemoglobinopathies

Synthesis of Hemoglobin

Hemoglobin synthesis
Heme synthesis starts with the condensation of glycine and succinyl coenzyme A under the action of a rate limiting enzyme daminolevulinic acid synthase. d-ALA will be formed. Pyridoxal phosphate (vit. B6) is a coenzyme for this reaction.

Hemoglobin synthesis
A series of biochemical reactions will follow. Two molecules of d-ALA condense to form a pyrrole called porphobilinogen (PBG) Four PBG condense to form a tetrapyrrole uroporphyrinogen III. UPG III is then converted to coproporphyrinogen.

Hemoglobin synthesis
CPG then changes to protoporphyrin IX which ultimately combines with iron in the ferrous state (Fe2+) to form heme. Iron is brought to the developing red cells by a carrier protein ( transferrin) which attaches to special binding sites on the surface of these cells. Transferrin releases iron and returns back to circulation.

Hemoglobin synthesis
Each molecule of heme combines with a globin chain. A tetramer of four globin chains each with its own heme group in a pocket is formed to make up a hemoglobin molecule.

Hemoglobin Synthesis: Heme

1 Aminolevulinate (ALA) synthase 2 ALA dehydratase 3 Uroporphyrinogen I synthase & Uroporphyrinogen III cosynthase 4 Uroporphyrinogen decarboxylase 5 Coproporphyrinogen III oxidase 6 Protoporphyrinogen IX oxidase 7 Ferrochelatase

Disorders of heme synthesis are called porphyrias.

Graphic accessed http://library.med.utah.edu/NetBiochem/images/pathway.gif, 2001.

An Overview of Iron Metabolism

Low pH of stomach solubilizes Fe-containing ionic compounds. Fe transporters facilitate absorption into blood stream Fe3+ ions are bound and chelated by Transferrin (Tf). Transferrin transports Fe to tissues Maintains solubility Keeps Fe ions unreactive Transferrin endocytosis is receptor-mediated (TfR) Endocytosis results in Fe3+ release Fe is distributed to topologically distinct regions of the cell via Fe transporter and/or channels (?) Usage: Protein components (Heme) Storage: Ferritin (Fe2+) Toxicity

Cells

Blood

Gut

Roles of Iron in the Cell

Fe(III)2-Tf Tf

Transferrin Receptors (TfR)

Proteins: Catalysis Electron, oxygen transport [Fe] Structural stabilization Sensor of Fe, ROS Formation of protein-bound radicals

[Fe]

Storage and Sequestration: Ferritin

[Fe] Toxicity: Oxidative stress

STRUCTURE OF HEME

Ferrous iron (Fe2+) Protoporphyrin IX: contains 4 pyrrole rings linked together by methenyl bridges

Hemoglobin Assembly

Graphic accessed http://evolvels.elsevier.com/section/default.asp?id=1138_ccalvo7_0001, 2008.

REMNANTS OF HEME PRODUCTION -normal excess porphyrin complexed to zinc= Free Erythrocyte Protoporphyrin -elevated when iron supply is diminished -ferritin- in cytoplasm, storage iron that was not used -hemosiderin

Disorders of Heme Synthesis

Acquired: Lead poisoning Congenital: Porphyrias Deficiency of heme has far-reaching effects (hemoglobin, cytochromes, etc.)

LEAD TOXICITY
Symptoms
Irritability Lethargy Sleeplessness Headaches Poor appetite Abdominal pain (with or without vomiting) Constipation

Pathophysiology
Binds to any compound with a sulfhydryl group Inhibits multiple enzyme reactions including those involved in heme biosynthesis (PBG synthase & ferrochelatase) One symptom of lead toxicity is increases in 5-ALA without concomitant increases in PBG

PORPHYRIAS
A group of rare disorders caused by deficiencies of enzymes of the heme biosynthetic pathway

The majority of the porphyrias are inherited in an autosomal dominant fashion thus, affected individuals have 50% normal levels of the enzymes, and can still synthesize some heme
Affected individuals have an accumulation of heme precursors (porphyrins), which are toxic at high concentrations Attacks of the disease are triggered by certain drugs, chemicals, and foods, and also by exposure to sun Treatment involves administration of hemin, which provides negative feedback for the heme biosynthetic pathway, and therefore, prevents accumulation of heme precursors

Hemoglobin Function
Each heme-globin unit has the ability to bind one molecular oxygen molecule Binding of oxygen requires cooperative movements by the rigidly arranged globin chains Oxygen affinity is also influenced by other factors

Graphic accessed http://www.monroecc.edu/depts/pstc/heme.gif, 2001.

Hemoglobin Function
Hemoglobin must be able to bind oxygen in the lungs but release it in the tissues An oxygen dissociation curve illustrates affinity of oxygen for hemoglobin under various conditions
Partial pressure of oxygen/carbon dioxide Concentration of 2,3-DPG Concentration of hydrogen ions Temperature Concentration of fetal hemoglobin Abnormal Hb variants

Graphic accessed http://www1.shore.net/~straub/labsk_hgbdis.gif, 2001.

Oxygen-hemoglobin dissociation curve

O2 carrying capacity of Hb at different Po2
Sigmoid shape
Binding of one molecule facilitate the second molecule binding Arterial blood- 95% = 95mmHg Venous blood- 70%= 40 mmHg P 50 (partial pressure of O2 at which Hb is half saturated with O2) 26.6mmHg

Hb-oxygen dissociation curve

Hb-oxygen dissociation curve

Right shift (easy oxygen delivery)
High 2,3-DPG High H+ High CO2 HbS

Left shift (give up oxygen less readily)

Low 2,3-DPG HbF

The Bohr Effect

Is the effect pH has on HGB-oxygen affinity
Increase pH shift to left (increased HGB affinity for O2)
HYPERVENTILATION

Decrease pH shift to right (decreased HGB affinity for O2 )

EXERCISE RENAL FAILURE

Graphic accessed http://www.med-ed.virginia.edu/courses/path/innes/images/nhgifs/hemoglobin2.gif, 2007.

Hemoglobin Function
In non-oxygenated hemoglobin, beta chains are farther apart 2,3-DPG forms salt bridges between the beta chains 2,3-DPG binding results in improved oxygen delivery to tissues

Tense form of hemoglobin

Hemoglobin Function
In oxygenated hemoglobin, beta chains are closer together 2,3-DPG salt bridges between the beta chains are sequentially broken The expelled 2,3-DPG results in increased oxygen affinity

Relaxed form of hemoglobin

Oxy & deoxyhaemoglobin

Hemoglobin Derivatives
Hemoglobin Name Methemoglobin Defect Oxidized Iron Blood Concetration Formation Iron Oxidation State 0.5-3.0% reversible Ferric reversible irreversible Ferrous Ferrous
(bluish skin discoloration= cyanosis); choc. brown bld; inherited/acquired, Hb M; trx: Vit. C, methylene blue

Carboxyhemoglobin CO-bound HGB <1% light sensitive with typical, brilliant, cherry red bld Sulfhemoglobin Sulfur-bound HGB <0.1%

sulfur in heme ring; mauve-lavender bld; carboxysulfhemoglobinemia; irreversible

By definition, a hemoglobin derivative differs from normal hemoglobin only by the molecule that occupies the space where oxygen binds. The resulting hemoglobin variant is NON-functional.

Reference Ranges Hemoglobin

Adult male Adult female Newborn Child 14 18 g/dL 12 16 g/dL 11 16 g/dL 12 15 g/dL