16Th March 2020 Blood Revised

Blood is the fluid circulating in a closed system of blood vessels and the chambers of the heart It is the medium which transports substances from one part of the body to the other Blood is composed of Plasma Platelets Cells WBCs RBCs (Erythrocytes) Hemoglobin (Hb) is red , oxygen carrying pigment present exclusively in erythrocytes HEMOGLOBIN A conjugated protein containing Globin Protein part ( 4 polypeptide chains- ) 96% of the total Hb mass Varies from species to species( species specificity) Heme Non protein (prosthetic group) Red colour Iron containing tetrapyrrole porphyrin derivative 4% of the total Hb mass Reversibly binds Oxygen Structure of Heme An Iron –porphyrin (Protoporphrin IX) compound with tetrapyrrole structure Protoporphyrin IX consists of 4 pyrrole rings combined through — CH= bridges (methyne bridges) The methyne bridges are referred as α,β,γ, and δ. The 2 Hydrogen atoms in the –NH groups pyrrole rings (II & IV) are replaced by Ferrous( Fe++) . The four pyrrole rings present in the porphyrin molecule are designated as I,II,III & IV . Each of these four rings has 2 groups attached to them M = Methyl –CH3 V = Vinyl – CH=CH2 P = Propionyl - CH2 - CH2 - COOH . The Fe++ can form 2 additional bonds .One of these position is linked internally (5th linkage ) to nitrogen of imidazole ring of Histidine of the Globin polypeptide chains . Other position is available to bind Oxygen Heme is the most prevalent metalloporphyrin in humans Common prosthetic group in Hemoglobin — Transport of O2 in blood Myoglobin — Storage of O2 in muscles Cytochromes — Part of electron transport chain Catalase — Degradation of H2O2 Tryptophan pyrolase — Oxidation of Tryptophan Cytochrome P450 — Hydroxylation of Xenobiotics HEME SYNTHESIS Major sites Liver Erythrocyte producing cells of bone marrow Rate of heme synthesis in liver is highly variable & depends upon size of heme pool while it is relatively constant in in bone marrow is relatively constant Mature RBC lack mitochondria and are unable to synthesize heme. Initial & last 3 reactions occur in mitochondria Intermediate steps takes place in cytosol 1. Formation of δ-amino levulinic acid Glycine (non- essential A.A) Succinyl CoA (intermediate in citric acid cycle) Glycine & succinyl CoA condense to form ALA- δ Amino levulinic acid . Enzyme is ALA synthase . Co-enzyme is pyridoxal PO4 . An intermediate compound α-amino β-keto- adipic acid is formed which on losing CO2 gives rise to ALA . ALA synthase activity is the rate-limiting factor in the synthesis of porphyrins ALA Synthase activity is increased by Barbiturates Sulfonylureas & decreased by Heme/ hemin High carbohydrate diet End product inhibition by hemin: When porphyrin production exceeds the availability of globin or other apoproteins heme accumulates & is converted to hemin by oxidation of Fe2+ to Fe3+ Hemin decreases the activity of ALA synthase by decreasing the synthesis of enzyme, through inhibition of mRNA synthesis. Effect of drugs on ALA synthase Administration of certain drugs increase hepatic ALA synthase activity eg. Griseofulvin – antifungal . Hydantions . Phenobarbitols- (anti convulsants used in t/m of epilepsy) These drugs are metabolized by microsomal cytochrome p450 monooxygenase system – a hemprotein oxidase system found in liver. In response to these drugs synthesis of cytochrome p450 increases leading to an increased consumption of heme - a component of cytochrome p450 This in turn decreases the heme conc in liver cells , leading to synthesis of ALA synthase resulting in increase in ALA synthesis. 2.Formation of porphobilinogen The condensation of 2 molecules of ALA takes place to give rise to porphobilinogen- mono pyrrole This reaction takes place in cytosol. o Enzyme – ALA dehydratase ( Zinc containing enzyme ) oIt needs reduced glutathione oALA dehydratase is extremely sensitive to inhibition by heavy metal ions like lead oThis inhibition is responsible for the elevated ALA and anemia seen in lead poisoning 3.Formation of hydroxymethylbilane . Porphobilinogen is converted to hydroxy methylbilane by the cytosolic enzyme uroporphyrinogen I synthase . Hydroxy methyl bilane is linear tetrapyrrole 4.Formation of uroporphyrinogen III Hydroxymethylbilane can spontaneously change into uroporphyrinogin I Uroporphyrinogin is the first cyclic tetra pyrrole derivative Hydroxymethylbilane is converted in to uroporphyrinogen III Enzyme –Uroporphyrinogen co-synthase Porphyrinogens are colourless compounds In porphyrinogens four pyrrole rings are attached to each other by methylene (-CH2-) bridges & N of each pyrrole ring has one H attached Porphyrinogens are readily converted by auto-oxidation to their respective porphyrins which are coloured compounds 5.Formation of coproporphyrinogen III . Occurs in cytosol . Acetate groups of uroporphyrinogen III lose CO2 & are changed to methyl groups . Enzyme – Uroporphyrinogen III decarboxylase 6.Formation of protoporphyrinogen IX Coproporphyrinogen III enters the mitochondria & its 2 propionate side chains are decarboxylated to vinyl groups forming protophophyrinogin IX Enzyme : Coprophyrinogen III oxidase oxidation & decarboxylation of 2 propionate chains 7.Formation of protoporphyrin IX Enzyme: Protoporphyrinogen IX oxidase. 8.Formation of Heme Introduction of iron into protoporphyrin occurs spontaneously but rate is increased by enzyme ferochelatase Reduced glutathione is needed for this reaction Enzyme – Ferochelatase This enzyme is inhibited by lead Heme containing Proteins Myoglobin Hemoglobin Myoglobin A heme protein present in heart & skeletal muscles functions as A reservoir for oxygen An oxygen carrier that increase the rate of transport of oxygen within the muscle cell Myoglobin consists of a single polypeptide chain 1. α Helical Content Myoglobin is a compact molecule with approximately 80% of its polypeptide chains folded into 8 stretches of α-Helix labeled as A to H. These α helix regions are terminated by The presence of Proline whose 5- membered ring can not be accommodated in an α helix or β-bends and loops stabilized by hydrogen bonds and ionic bonds Location of polar and non polar Amino acid residues Interior consists almost entirely of nonpolar residues Exterior consists of charged, polar and nonpolar residues Histidine is found in the interior of the protein which play a critical role in the binding of iron and oxygen The polar amino acids can form hydrogen bonds with each other and with water Binding of the heme groups The heme group of myoglobin sits in crevice in the molecule which is lined with non polar amino acids Two histidine residues are very important . Proximal histidine (F helix) binds directly to the iron of heme . Distal histidine (E helix) helps to stabilize the binding of oxygen to the ferrous iron Molecular wt. — 17,000 Combines with O2 to form oxymyoglobin & serves as O 2 reservoir within muscle cells The O2 binding power of myoglobin is much more so oxygenated myoglobin cannot readily provide its O2 to tissue When pO2 of tissues falls to 5 mm of Hg or below oxy Mb dissociates to provide O2 to tissue mitochondria for ATP formation. HAEMOGLOBIN Haemoglobin — a conjugated protein Heme — prosthetic group Globin — apo protein part 4 polypeptide chains of 2 different monomeric units ( α and β chains ) Each α chain contains 141 amino acids Each β chains 146 amino acids Each sub unit contains a heme group Found exclusively in RBCs Mol. wt.— 64458 Main function is to transport O2 from lungs to the tissues transport of CO2 from tissues to lungs Hb A Major adult Hb is composed of four polypeptide chains 2 α chains 2 β chains These 4 chains are held together by non- covalent interactions. Each subunit has stretches of α-helical structure & a heme binding pocket similar to that of myoglobin The tetrameric Hb molecule is structurally & functionally more complex than myoglobin + Hb can transport H and CO2 from tissues to lungs and can carry 4 molecules of O2 from the lungs to cells of the body . Oxygen binding properties of Hb are regulated by interaction with allosteric effectors Quaternary structure of Hb The Hb tetramer is composed of 2 identical diamers (α β)1 and (α β)2 The two polypeptide chains in each diamer are held tightly together primarily by hydrophobic interactions . Hydrophobic amino acid residues are localized in the interior & also on the surface of the each subunit . Interchain hydrophobic interactions form strong associations between α and β sub units in the diamers . Ionic and H+ bonds also occur between the members of a diamer The two diamers are able to move with respect to each other being held together primarily by polar bonds The weaker interactions between these mobile diamers result in the 2 diamers occupying different relative positions in deoxy Hb as compared with oxy Hb The binding of O2 to heme iron pulls the iron into the plane of heme The iron is also linked to the proximal histidine (F8), there is movement of globin chains that alters the interface between α β dimers Hemoglobin exists in 2 forms T form ( Taut /Tense form) R form ( Relaxed form) T form The deoxy from of Hb is called the T or Taut (Tense) form In T form the two α β diamers interact through a network of ionic bonds & H+ bonds that constrain the movement of polypeptide chains The T form is the low O2 affinity form of Hb R form The binding of O2 to Hb causes the rupture of some of the ionic bonds & H+ bonds between α β diamers This leads to a structure called R or relaxed form in which polypeptide chains have more freedom of movement

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