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Hemoproteins
Consist of hem– cyclic tetrapyrrole– 1 iron cation Fe2+ bound
in the middle of tetrapyrrole scelet by coordination covalent bonds
– conjugated system of double bonds
methine bridge
pyrrole ring
Types of hemoglobin
Adult HbA: 2α and 2β subunits (98%HbA)
Adult HbA2: 2α and 2δ subunits (2% HbA)
Fetal HbF: 2α and 2γhave higher O2 affinity than HbA – take up oxygen from
the maternal circulation
Embryoinic: 2and 2 2 and 2
2 and 2
have higher O2 affinity than HbA
Hemoproteins
Hemoglobin (transports O2 to the tissues) Myoglobin (stores O2 in the muscles)
Cytochromes (e- carriers in ETC) Catalase + peroxidases (decomposition of peroxides) Cytochrome P-450 (hydroxylation) Desaturasases FA (desaturation FA)
Redox state Fe 2+ Fe 3+
Redox state Fe 2+
Structure of Hemoglobin • 4 polypetide subunits (globins)• Hb A (adults) heterotetramer 2α a 2β• Each subunit contains 1 hem group• 8 helices (A-H) β subunit• 7 helices α subunit• Hydrofobic pocket
- protect hem against oxidation
• Hem binding to globin– Fe 2+ is coordinated by N atom from proximal histidin F8
• Binding of O2 – distal histidin E7 hydrogen bonds to the O2
Structure of Hemoglobin
• Quaternary structureInteractions between subunits
1) hydrofobic ( between α-β)2) electrostatic (between α-α; β-β, α-β)
– O2 binding – loss of these interactions
Structure of Hemoglobin
α1
α2
β1β2
• 1 polypeptide chain (153 AA)• 1 heme• Tertiary structures of the α and β subunits are remarkably similar, both to
each other and to that of Mb• Skeletal and heart muscles
Structure of Myoglobin
Binding of O2 (oxygenation)
• Oxygenation changes the electronic state of the Fe2+ - heme• Color change of blood from dark purplish (venous) to the brilliant scarlet
color (arterial)
• The binding of the first O2 to Hb enhances the binding futher O2 molecules
• O2 affinity of Hb increases with increasing pO2
• Sigmoidal saturation curve
• Hyperbolic curve for Mb - no cooperative behavior
Mechanism of oxygen-binding cooperativity
Satu
ratio
n O
2
• Hb loads O2 to about 90% saturation under the arterial partial pressure
• Hb travels to the tissue where the O2 partial pressure is 20 torr, most of Hb´s bound O2 is released
• The diference in oxygen affinity between Mb and Hb is greatest between 5 and 30 torr, where Mb binds much more O2 than does Hb. This difference allows O2 to be released at the tissues from O2 - loaded Hb, and transported to Mb Sa
tura
tion
O2
• The movement of Fe 2+ into the heme plane triggers the T→R conformational shift
•The loss of electrostatic interactions induce conformational changes in all other subunits
Conversion of T form→R form
T form (tense) R form (relaxed)
The binding of the first O2 molecule to subunit of the T-form leads to a local conformational change that weakens association between the subunits R-form
Allosteric effectors
• CO2 • H+
• 2,3-bisphosphoglycerate
Decrease O2 affinity of Hb
Influence the equilibrium between T and R forms
2,3 - bisphosphoglycerate
• binds selectively to deoxy-Hb• stabilizes T form • lowers the affinity of Hb for oxygen • oxygen is more readily released in tissues
2,3 - bisphosphoglycerateClinical aspects:
In people with high-altitude adaptation or smokers the concentration of 2,3-BPG in the blood is increased increases the amount of oxygen that Hb unloads in the capilaries
Fetal hemoglobin (HbF α2γ2), has low BPG affinity – the higher O2 affinity – facilitates the transfer of O2 to the fetus via the placenta
Bohr effect• The binding of protons H+ by Hb lowers its affinity for O2
• Increasing pH, that is, removing protons,stimulates Hb to bind O2
• pH of the blood decreases as it enters tissues because CO2 produced by
metabolism is converted to H2CO3
• Dissociation of H2CO3 produces protons• Promote the release of oxygen
In the tissues
Bohr effect
In the lungs
Oxygen binds to Hb, causing a release protons, which combine with bicarbonate to form H2CO3
Carbonic anhydrase cleaves H2CO3 to H2O and CO2
CO2 is exhaled
Total Hb and Free Hb
• Reference values of total Hb – age and sex dependent, about 150 g/l
• Free Hb: 125 – 300 mg/l
Derivatives of hemoglobin
Deoxyhemoglobin – Hb without O2
Oxyhemoglobin – Hb with O2
Carbaminohemoglobin – Hb with CO2
– CO2 is bound to globin chain
– about 15% of CO2 is transported in blood bound to Hb
Carbonylhemoglobin – Hb with CO – CO binds to Fe2+ 200x higher affinity to Fe2+ than
O2 – poisoning, smoking
Methemoglobin – (metHb) contains Fe3+ instead of Fe2+
Autooxidation of hemoglobin
3% of hemoglobin undergoes oxidation every day
Hem – Fe2+- O2 Hem - Fe3+ + O2•-
Methemoglobin reductase
reduces methemoglobin
FAD, cytochrom b5 a NADH
Methemoglobinemia
1. Hereditary deficit of methemoglobin reductase
2. Abnormal hemoglobin HbM (Hb mutation)
3. Exposure to exogenous oxidizing drugs (sulfonamides, aniline)
Clinical aspects: cyanosis (10% Hb forms metHb)treatment: administration of methylene blue or ascorbic acid
Glycohemoglobin (HbA1c)
Formed by Hb‘s exposure to high levels of glucose
Nonenzymatic glycation of terminal NH2 group (Val) β-chain
Normally about 4 % of Hb is glycated (proportional to blood Glc concentration)
People with DM have more HbA1c than normal ( 5%)
Measurement of blood HbA1c is useful to get information about long-term control of glycemia
β1
β2
α1
α2
α1
α2
α1
α2
γ1
γ2
δ1
δ2
Hb A > 96,5% Hb F < 1% Hb A2 < 3,5%
HbA1c: What are we looking for?
β1
β2
α1
α2
Heme
Heme1st Step: Unstable, reversible reaction between Glucose and the N-terminal valine of the β-chain (Schiff base)
Labile Hb A1c
2nd Step: During red blood cell circulation, some of the labile A1C is converted to form a stable HbA1c (Amadori rearrangement)
Non-Enzymatic Glycation of Hb A
Stable Hb A1c
HbA1c is currently defined as:
Hemoglobin A which is irreversibly glycated at one or both N-terminal Valines of the chains in the tetramer.Glycation elsewhere on the or chains is irrelevant.
G
G
G
G
G
G
G
G
G
N
N
N
N
N
All of these are HbA1c
The nature of the problem – what is HbA1c?
Glycohemoglobin, or GHb, or Total GHb, is defined as:
Hb having one or more sugars irreversibly attached at any point in any of the globin chains.
(This also includes all forms of HbA1c).
G
G
N
G
G
GN
G
N
G
GG
GN
All of these are GHb (but not HbA1c)
The nature of the problem – what is HbA1c?
Hb A093-95%
Hb A1 = GHbGlycated Hbs
5-7%
Hb A
+ + +
Hb A1a0,5%
Fructose-1,6-diphosphateGlucose-6-phosphate
Hb A1b0,5%
pyruvate
Hb A1c4-6%
glucose
Hb FHb A2
HbA1c: What are we looking for?
Glycation at theN-terminal Valin
of the β-globin chain
The Pros and the Cons of using HbA1c for Diabetes DiagnosisDavid B.Sacks; AACC Webinar April 10th 2012
Hemoglobinopathies
mutation → abnormal structure of the hemoglobin Large number of haemoglobin mutations, a fraction has deleterious effects:
sickling, change in O2 affinity, heme loss or dissociation of tetramer hemoglobin M and S, thalassemias
1. Hemoglobin M• Replacement of His E7α by Tyr (Hb Boston) or• Replacement of Val E11β by Glu (Hb Milwaukee) • the iron in the heme group is in the Fe3+ state (methemoglobin) stabilized by
the tyrosine or by glutamate • Methemoglobin reductase cannot reduce Fe3+ • methemoglobin can not bind oxygen
2. Thalassemias• Mutation that results in decreased synthesis of α or β-chains• thalassemia mutations provide resistence to malaria in the
heterozygous state
α- thalassemias – complete gene deletion
4 α globin genes per cell: 1 copy of gen is deleted: without symptoms 2 copies are deleted: RBC are of decreased size (microcytic) and reduced Hb
concentration (hypochromic), individual is usually not anemic 3 copies are deleted: moderately severe microcytic hypochromic anemia with
splenomegaly 4 copies are deleted: hydrops fetalis: fatal in utero
Excess β chains form homotetramer HbH which is useless for delivering oxygen to the tissues (high oxygen affinity)
• β+ – some globin chain synthesis• β0 – no globin chain synthesis
Heterozygotes: microcytic hypochromic RBC, mild anemia
Homozygotes β0 β0 : severe anemia
Excess α chains precipitate in erythroid precursor – their destruction-ineffective erythropoiesis
β- thalassemias
3. Hemoglobin S (sickle-cell)
• Causes a sickle-cell anemia
• Replacing Glu A3β with the less polar amino acid Val - forming „an adhesive region“ of the β chain• HbS proteins aggregate into a long rodlike helical fiber
Sickle-cell anemia
Red blood cells adopt a sickle shape in a consequence of the forming haemoglobin S fibersThe high incidence of sickle-cell disease coincides with a high incidence of malariaIndividuals heterozygous in HbS have a higher resistance to malaria; the malarial parasite spends a portion of its life cycle in red cells, and the increased fragility of the sickled cells tends to interrupt this cycle