Hemoglobin and hemoglobinpathies Srbová M., Průša R.

Hemoproteins Consist of hem – cyclic tetrapyrrole – 1 iron cation Fe 2+ 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 O 2 affinity than HbA – take up oxygen from the maternal circulation Embryoinic: 2  and 2  2  and 2  2  and 2  have higher O 2 affinity than HbA

Hemoglobin switching Alteration of globin gene expresion during development

Hemoproteins  Hemoglobin (transports O 2 to the tissues)  Myoglobin (stores O 2 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 O 2 – distal histidin E7 hydrogen bonds to the O 2 Structure of Hemoglobin

Quaternary structure Interactions between subunits 1) hydrofobic ( between α-β) 2) electrostatic (between α-α; β-β, α-β) – O 2 binding – loss of these interactions Structure of Hemoglobin α1α1 α1α1 α2α2 α2α2 β1β1 β1β1 β2β2 β2β2

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 O 2 (oxygenation) Oxygenation changes the electronic state of the Fe 2+ - heme Color change of blood from dark purplish (venous) to the brilliant scarlet color (arterial)

The binding of the first O 2 to Hb enhances the binding futher O 2 molecules O 2 affinity of Hb increases with increasing pO 2 Sigmoidal saturation curve Hyperbolic curve for Mb - no cooperative behavior Mechanism of oxygen-binding cooperativity

Saturation O 2 Hb loads O 2 to about 90% saturation under the arterial partial pressure Hb travels to the tissue where the O 2 partial pressure is 20 torr, most of Hb´s bound O 2 is released

Saturation O 2

The diference in oxygen affinity between Mb and Hb is greatest between 5 and 30 torr, where Mb binds much more O 2 than does Hb. This difference allows O 2 to be released at the tissues from O 2 - loaded Hb, and transported to Mb Saturation O 2

Oxygen binding to Hb

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 O 2 molecule to subunit of the T-form leads to a local conformational change that weakens association between the subunits  R-form

Allosteric effectors CO 2 H + 2,3-bisphosphoglycerate Decrease O 2 affinity of Hb Influence the equilibrium between T and R forms

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 α 2 γ 2 ), has low BPG affinity – the higher O 2 affinity – facilitates the transfer of O 2 to the fetus via the placenta

Bohr effect The binding of protons H + by Hb lowers its affinity for O 2 Increasing pH, that is, removing protons,stimulates Hb to bind O 2 pH of the blood decreases as it enters tissues because CO 2 produced by metabolism is converted to H 2 CO 3 Dissociation of H 2 CO 3 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 H 2 CO 3 Carbonic anhydrase cleaves H 2 CO 3 to H 2 O and CO 2 CO 2 is exhaled

Hemoglobin and transport CO 2

Hemoglobin determination 2. Direct spectrophotometry of plasma 415 – 460 nm 1.

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 O 2  Oxyhemoglobin – Hb with O 2  Carbaminohemoglobin – Hb with CO 2 – CO 2 is bound to globin chain – about 15% of CO 2 is transported in blood bound to Hb  Carbonylhemoglobin – Hb with CO – CO binds to Fe x higher affinity to Fe 2+ than O 2 – poisoning, smoking

 Methemoglobin – (metHb) contains Fe 3+ instead of Fe 2+ Autooxidation of hemoglobin 3% of hemoglobin undergoes oxidation every day Hem – Fe 2+ - O 2 Hem - Fe 3+ + O 2 - Methemoglobin reductase reduces methemoglobin FAD, cytochrom b 5 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 (HbA 1c )  Formed by Hb‘s exposure to high levels of glucose  Nonenzymatic glycation of terminal NH 2 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 HbA 1c is useful to get information about long-term control of glycemia

β1β1 β2β2 α1α1 α2α2 α1α1 α2α2 α1α1 α2α2 γ1γ1 γ2γ2 δ1δ1 δ2δ2 Hb A > 96,5%Hb F < 1%Hb A2 < 3,5% HbA1c: What are we looking for?

1 st Step: Unstable, reversible reaction between Glucose and the N-terminal valine of the β-chain (Schiff base) 2 nd Step: During red blood cell circulation, some of the labile A1C is converted to form a stable HbA1c (Amadori rearrangement)

HbA 1c 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.   GG   GG GG   GG GG   GG GG GG GG NN NN NN NN NN All of these are HbA 1c The nature of the problem – what is HbA 1c ?

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 HbA 1c ).   GG   GG NN   GG GG GG NN GG NN   GG GG GG GG NN All of these are GHb (but not HbA 1c ) The nature of the problem – what is HbA1c?

Hb A % Hb A1 = GHb Glycated Hbs 5-7% Hb A + ++ Hb A1a 0,5%Fructose-1,6-diphosphateGlucose-6-phosphate Hb A1b 0,5%pyruvate Hb A1c 4-6%glucose Hb F Hb A2 HbA1c: What are we looking for? Glycation at the N-terminal Valin of the β-globin chain

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 O 2 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 Fe 3+ state (methemoglobin) stabilized by the tyrosine or by glutamate Methemoglobin reductase cannot reduce Fe 3+ 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 β 0 – no globin chain synthesis Heterozygotes: microcytic hypochromic RBC, mild anemia Homozygotes β 0 β 0 : 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 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)