Biochemistry_of_Blood_Part_II_Hemoglobin_Structure_FA23_LEO.pptx

BIOCHEMISTRY OF BLOOD-HEMOGLOBIN Vanessa De La Rosa, PhD CV I FA 2023 1 SESSION OBJECTIVES • Predict how allosteric regulation by temperature, H+, BPG, and CO2 effect the P50 of hemoglobin • Explain the role of hemoglobin in the delivery of oxygen, removal of waste and buffering • Describe the st...

BIOCHEMISTRY OF BLOOD-HEMOGLOBIN Vanessa De La Rosa, PhD CV I FA 2023 1 SESSION OBJECTIVES • Predict how allosteric regulation by temperature, H+, BPG, and CO2 effect the P50 of hemoglobin • Explain the role of hemoglobin in the delivery of oxygen, removal of waste and buffering • Describe the structural basis for cooperativity of oxygen binding to hemoglobin • Explain the effects of carbon monoxide on hemoglobin function • Describe the biochemical significance of acquired hemoglobin variants, such as hemoglobin A1C, methemoglobin, and cyanmethemoglobin. • Relate hemoglobin function to glycolysis • Identify and compare the effects of the major 2 GLOBULAR PROTEINS Functions •Storage of ions and molecules  myoglobin, ferritin •Transport of ions and molecules  hemoglobin, serotonin transporter •Defense against pathogens  antibodies, cytokines •Muscle contraction  actin, myosin •Biological catalysis  chymotrypsin, lysozyme Surface-hydrophilic (gray) vs. core-hydrophobic (cyan) residues in a water-soluble protein. 3 GLOBULAR HEMEPROTEINS • • Contain heme group Ferrous iron (Fe2++) can form 6 bonds 2 bonds on each side of the planar porphyrin ring  Hemoglobin  Myoglobin  CYP450  Catalase Both bind oxygen reversibly 4 MYOGLOBIN & HEMOGLOBIN STRUCTURE Myoglobin Hemoglobin Heart and skeletal muscle Exclusively in red blood cells Single chain, similar to beta globin chain Tetramer of 2 identical dimers Oxygen storage Non-polar amino acids line interior, charged AAs exclusively on surface Strong interaction within each dimer via hydrophobic (HbA) interactions α1 (αβ β1 )1 Weak ionic and hydrogen bonding between dimer pairs Oxygen transport Non-polar AAs line interior and localized regions on exterior β2 (αβ)2α2 5 GLOBIN GENE SWITCHING Globin gene switching produces a variety of different hemoglobin tetramers throughout development Switching is controlled primarily at the transcriptional level 6 HbF MAJOR HEMOGLOBINS •major form during fetal α γ •two adult α -subunits γ α •two fetal β-subunits development 2 HbA •major form shortly after birth through adulthood •two adult α2-subunits •two adult β-subunits 7 HEMOGLOBIN FUNCTION • Transports oxygen from region of high concentration (lungs) to peripheral tissues where oxygen tension is low • Transports CO2 and H+ (generated by metabolism in peripheral tissues) back to lungs 8 OXYGEN BINDING TO MYOGLOBIN • α- helical secondary and tertiary structure • Binds one oxygen molecule • Oxygen binds to ferrous iron atom on heme group • The oxygen binding curve is hyperbolic (more on this) • Absence of allosteric interactions 9 HEMOGLOBIN EXHIBITS SEQUENTIAL COOPERATIVITY FOR OXYGEN BINDING binding of oxygen to one subunit induces conformational change that is partially transmitted to adjacent subunits  transmission of partial conformational change increases affinity for oxygen by adjacent subunits See website link for additional 3D interactive images DeoxyHb T (tense) state Low affinity movement of iron mediated by proximal histidine OxyHb R (relaxed) state High affinity 10 T AND R STATES OF HEMOGLOBIN T = Tense state  More interactions, more rigid  Lower affinity for O2 R = Relaxed state,  Fewer Interactions, more flexible  Higher affinity for O2 O2 binding triggers a T  R conformational change Conformational change from the T state to the R state involves breaking ion pairs between the dimer pair interface 11 STRUCTURE FUNCTION RELATIONSHIPS Myoglobin exhibits hyperbolic binding •due to simple monomeric structure •when oxygen becomes limiting during exercise, myoglobin releases oxygen to help maintain high level of activity for longer period of time Hemoglobin exhibits sigmoidal cooperative binding •due to complex tetrameric structure •critical for efficiency in loading oxygen in lungs and unloading in periphery •P50 is partial pressure of oxygen yielding 50% saturation of Hb •lower P50 = higher oxygen affinity 12 HEMOGLOBIN FORMS DIFFER IN THEIR BINDING AFFINITY FOR OXYGEN HbF has higher affinity for oxygen than does HbA • facilitates dissociation from HbA & subsequent binding by HbF at oxygen concentrations found at placental interface leftward shift in oxygen dissociation curve 13 ALLOSTERIC REGULATION OF OXYGEN BINDING Allosteric regulation in hemoglobin involves several effector molecules decrease oxygen affinity for hemoglobin (shift curve to right) H+ & CO2 are negative allosteric effectors of oxygen binding •enhance efficiency of oxygen unloading in peripheral tissues •facilitate H+ & CO2 transport from peripheral tissues to lungs 14 ALLOSTERIC REGULATION OF OXYGEN BINDING Bohr Low pH or high CO2= shift right effect High pH or low CO2= shift left Deoxy Hb H+ binding results from shift in pKa of specific residues (mostly histidines) Some CO2 binds to Nterminal lysine residues in Hb - helps stabilize T-state Source of protons; lower pH Become protonated, and form bonds that stabilize T state 15 ALLOSTERIC REGULATION OF OXYGEN BINDING Bohr Low pH or high CO2= shift right effect High pH or low CO2= shift left Deoxy Hb H+ binding results from shift in pKa of specific residues (mostly histidines) Some CO2 binds to Nterminal lysine residues in Hb - helps stabilize T-state Become protonated, and form bonds that stabilize T state 16 ALLOSTERIC REGULATION OF OXYGEN BINDING 2,3-bisphosphoglycerate (2,3-BPG or DPG) is a negative allosteric effector of oxygen binding DeoxyHb • regulatory mechanisms in red cells control 2,3-BPG concentration: • fine tunes oxygen affinity for hemoglobin in response to changes in metabolism & environment, e.g., high altitude • obviates need to induce binds to central cavity between β subunits (dimer pairs) through ionic interactions 17 ALLOSTERIC REGULATION OF OXYGEN BINDING 2,3-bisphosphoglycerate (2,3-BPG or DPG) is a negative allosteric effector of oxygen binding • regulatory mechanisms in red cells control 2,3-BPG concentration: • fine tunes oxygen affinity for hemoglobin in response to changes in metabolism & environment, e.g., high altitude • obviates need to induce 18 REGULATION OF OXYGEN BINDING Temperature  temperature =  oxygen affinity Facilitates oxygen unloading during elevated metabolic rates associated with fever 19 CARBON MONOXIDE POISONING •CO has much higher affinity (>200-fold) for heme iron than oxygen •“Locks” hemoglobin in R state, causing high affinity for oxygen at remaining subunits •Inability to release oxygen to tissues 20 HEMOGLOBINOPATHIES Disorders caused by abnormal hemoglobins- Review summary handout on LEO Sickle Cell Production of structurally abnormal hemoglobin (HbS) Thalassemia Insufficient quantity of normal hemoglobin 21 SICKLE CELL ANEMIA (HbS) •Most common hemoglobinopathy •Homozygous recessive disease caused by point mutation in adult β-globin gene (βS allele) •Sickle cell anemia specific to homozygous HbS/HbS •GluVal substitution at position 6 in adult β-globin polypeptide •HbS has two normal adult α-globin subunits & two sickle adult βglobin subunits 22 SICKLE FIBERS HbS polymerizes forming intracellular fibers when deoxygenated critical contact made by β6 Val of one molecule & hydrophobic acceptor pocket of a β subunit in another molecule formed by Leu88 & Phe85 surrounded by hydrophilic residues Rate and extent of polymer formation depends on: • degree of deoxygenation • intracellular hemoglobin concentration • relative amount of HbF 23 SICKLING ERYTHROCYTE ABNORMALITIES Sickling results in: • Reduced erythrocyte deformability • Vaso-occlusion (defective passage through the microcirculation) • Chronic hemolytic anemia (shorter erythrocyte 1/2 life) • Dehydration • Abnormal membrane phospholipid asymmetry • HbC variant characterized by GluLys THALASSEMIAS Characterized by imbalances in relative concentrations of  -globin and β -globin chains  -thalassemia caused by decrease/deficiency in  -globin chains β-thalassemia caused by decrease/deficiency in β-globin chains caused by many different types of mutations in adult βglobin gene that affect expression 25 βTHALASSEMIA S •Caused by point mutations that affect the production of functional β-globin mRNA •α-globin chain synthesis is normal •α-globin chains precipitate, causing the premature death of cells initially destined to become mature RBC •Increase in HbA2 and HbF Gel electrophoresis Detects and quantifies (%) hemoglobin variants Detects abnormal hemoglobin 26 patterns αTHALASSEMIA S •Caused by deletion mutations Gel electrophoresis •Genome contains 4 copies of the α-globin gene •Several levels of α-globin chain deficiencies Compare βthalassemia vs. αthalassemia 27 ACQUIRED HEMOGLOBIN VARIANTS: HbA1c attached nonenzymatically to N-terminal valine of β-chain in HbA • Normally present in very low levels • Reduced affinity for 2,3-BPG • Quite abundant in diabetics, particularly those with poor glycemic control • Reflects long-term glycemic exposure (preceding 8-12 wks) Glycated hemoglobin • Preferred clinical index for diagnosing diabetes (≥ 6.5%) 28 METABOLI SM AND HbA FUNCTION Glycolysis and PPP critical for normal Hb function Methemoglobin (MetHb) is oxidized form of hemoglobin G6P D Methemoglo bin Does not bind oxygen Increase s with PK deficienc y NADH required for reduction of MetHB to HbA Pyruvate kinase (PK) 29 METHEMOGLOBINEMIA Inability to reduce metHb to HbA • inherited NADH cytochrome-b5 methemoglobin reductase deficiency • hemoglobin mutations (collectively called hemoglobin M variants) • exogenous oxidizing agents (e.g., nitrates in well water) Symptoms proportional to metHb concentration: • up to 15%; skin color changes (cyanosis with blue or grayish pigmentation) blood color changes (brown or chocolate color) • >15%; neurologic and cardiac symptoms due to hypoxia 30 Produced during treatment for cyanide (CN) poisoning • CN poisoning produces cytotoxic anoxia due to cytochrome oxidase inhibition • Treatment with nitrites (intravenous infusion of NaNO2 or inhalation of amyl nitrite) converts HbA to metHb • CN replaces OH at position 6 of iron producing cyanmetHb • CyanmetHb eliminated by normal processes CYANMETHEMOG LOBIN 31