Transcript Hemoglobin a hemoglobinpatie

Hemoglobin and
hemoglobinpathies
Srbová M., Průša R.
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 2γ
have higher O2 affinity than HbA – take up oxygen
from the
maternal circulation
Embryoinic:
2and 2
2 and 2 
2 and 2
have higher O2 affinity than HbA
Hemoglobin switching
Alteration of globin
gene expresion during
development
Hemoproteins
Redox state Fe 2+
 Hemoglobin (transports O2 to the tissues)
 Myoglobin (stores O2 in the muscles)
Redox state Fe 2+




Fe 3+
Cytochromes (e- carriers in ETC)
Catalase + peroxidases (decomposition of peroxides)
Cytochrome P-450 (hydroxylation)
Desaturasases FA (desaturation FA)
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
Structure of Hemoglobin
• 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 structure
Interactions between subunits
1) hydrofobic ( between α-β)
2) electrostatic (between α-α; β-β, α-β)
– O2 binding – loss of these interactions
α1
β2
β1
α2
Structure of Myoglobin
• 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
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)
Mechanism of oxygen-binding
cooperativity
• 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
• Hb travels to the
tissue where the O2
partial pressure is 20
torr, most of Hb´s
bound O2 is released
Saturation O2
•Hb loads O2 to
about 90%
saturation under the
arterial partial
pressure
Saturation O2
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
Saturation O2
•
Oxygen binding to Hb
•The loss of electrostatic interactions induce
conformational changes in all other subunits
• The movement of Fe 2+ into the heme plane triggers the T→R
conformational shift
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
Influence the equilibrium between T and R forms
• CO2
• H+
• 2,3-bisphosphoglycerate
Decrease O2 affinity of Hb
Oxygen transport regulation
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 - bisphosphoglycerate
Clinical 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
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
In the lungs
Hemoglobin and transport CO2
Hemoglobin determination
1.
2.
Direct spectrophotometry of plasma 415 – 460 nm
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
HbA1c: What are we looking for?
Hb A > 96,5%
Hb F < 1%
Hb A2 < 3,5%
β1 α1
γ1 α1
δ1
α1
β2
α2 γ2
α2 δ2
α2
st Step:
2
red blood
cell circulation,
some
of the labile
A1C isand
1nd
Step:During
Unstable,
reversible
reaction
between
Glucose
converted
to formvaline
a stableof
HbA1c
(Amadori
rearrangement)
the N-terminal
the β-chain
(Schiff
base)
The nature of the problem – what is HbA1c?
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
N






N
G
G
G
N


G



N

G
G
All of these are HbA1c
G
N




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
G




G
N




G
N
G
G
N
G
N




G
All of these are GHb (but not HbA1c)
G
HbA1c: What are we looking for?
Hb A
Hb A0
93-95%
Hb A1 = GHb
Glycation at the
N-terminal Valin
of the β-globin chain
Glycated Hbs
5-7%
Hb A1a Hb A1b Hb A1c
0,5% 0,5%
4-6%
Fructose-1,6-diphosphate
Glucose-6-phosphate
Hb A2
Hb F
+
pyruvate
+ +
glucose
The Pros and the Cons of using HbA1c for Diabetes Diagnosis
David B.Sacks; AACC Webinar April 10th 2012
Glycohemoglobin Assay
HPLC – TOSOH G7
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- thalassemias
• 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 destructionineffective erythropoiesis
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 fibers
The high incidence of sickle-cell disease coincides with a high incidence of malaria
Individuals 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
Pictures used in the presentation:
• Marks´ Basic Medical Biochemistry, A Clinical Approach, third
edition, 2009 (M. Lieberman, A.D. Marks)
• Principles of Biochemistry, 2008, (Voet D, Voet J.G., and Pratt
C.W)
• Color Atlas of Biochemistry, second edition, 2005 (J. Koolman
and K.H. Roehm)