Presented by Gyandeep Gupta

FPB-MA5-02

Submitted toDr.Sujata sahoo

FISH HEMOGLOBIN

INTRODUCTION• Haemoglobin iron containing oxygen transport

metalloprotein in the red blood cells .• Found in all vertebrates with the exception of the fish

family Channichthyidae.• discovered by Hunefeld in 1840.• Primary function of the RBCs -manufacture hemoglobin.• transports oxygen to the tissues and carbon dioxide from

tissues to the lungs.• composed of four subunits, each containing heme and

globin

STRUCTURE OF HEMOGLOBIN

• hemoglobin -most fishes is a tetrameric molecule• molecular weight- 60 000–70 000Da.• consists of four globin chains- two α- and hains.• globin chains have 140–160 amino acids (Mol.wt,15 000 and

17 000 Da).• serves to make the binding of oxygen to heme-iron reversible.• Heme is situated in a hydrophobic pocket of globin.

Structure of hemoglobin

• A major feature of teleost fish haemoglobins is their multiplicity.

• teleost fish have 4-5 histidines per globin chain, the other vertebrates including elasmobranch fish typically have double this amount .

• Fish haemoglobins characteristically have serine substituted for cysteine in the position 93 of the β-globin chain.

• Fish haemoglobins also have the end-terminal amino acid of the α-chain acetylated.

• iron performs its function in the ferrous state

STRUCTURE OF HEME

• Heme-derivative of the porphyrin.• produced by the combination of iron with a

porphyrin ring.• Prophyrins -cyclic compounds formed by fusion of

4 pyrrole ring linked by methenyl (=CH-) bridges.• The pyrrole rings-named as I, II, III, IV • bridges as α, β, ϒ and δ. • The possible areas of substitution-denoted as 1 to

8.

Biosynthesis of Heme

CATABOLISM OF HEME

• End product of heme catabolism-bile pigments.

• When old RBCs breaks down-liberate hemoglobin.

• Iron liberated from heme is re-utilized.• The porphyrin ring is broken down in reticulo-

endothelial cells of liver and spleen into bile pigments, mainly bilirubin.

Hb DIFFERENTION IN FISHESPolymorphism in hemoglobin-associated with the

level of activity of fish species.all animal hemoglobins share the same heme group.differences in their properties: – including O2 affinity– Electrophoretic mobility– pH sensitivity

Fish hemoglobin -two types viz; MonomericTetrameric

Agnatha – monomeric hemoglobin.tetrameric hemoglobin are also of many kinds Example:

4 kinds in rainbow trout.Gold fish- 3 Eel- 2

• Each type has different functional property.

DERIVATIVES OF Hb• Oxyhemoglobin (oxyHb) = Hb with o₂• Deoxyhemoglobin (deoxyHb) = Hb without o₂• Methemoglobin (metHb) = Fe3+ instead of Fe2+ in heme

groups• Carbonylhemoglobin (HbCO) = CO binds to Fe2+ in

heme in case of CO poisoning or smoking. (CO has 200x higher affinity to Fe2+ than O₂).• Carbaminohemoglobin (HbCO2) = CO₂is non-covalently

bound to globin chain of Hb. • HbCO₂ transports CO₂ in blood (about 23%).

TENSED AND RELAXED STATES OF Hb

exists in two major conformational states: Relaxed (R ) and Tense (T)

R state- higher affinity for O₂.In the absence of O₂, T state is more stable. But when O₂ binds to hemoglobin, it undergoes a

conformational change to the R state, which becomes more stable.

The structural change involves readjustment of interactions between subunits.

TRANSPORT OF OXYGEN

• In the gills at the lamellar –capillary interface, the partial pressure of oxygen is typically high, and therefore the oxygen binds readily to hemoglobin

• Hemoglobin releases the oxygen into the tissue due to lower oxygen partial pressures.

OXYGEN DISSOCIATION CURVE

• The curve between the percentage saturation of hemoglobin with oxygen (y-axis) and the partial pressure of oxygen in the blood (x-axis).

• Important tool for understanding how blood carries and releases oxygen.

• oxygen dissociation curve for oxyhaemoglobin is S/sigmoid-shape.

• It shows how the saturation of haemoglobin with oxygen varies with partial pressure of oxygen.

• At lower partial pressures, oxyhaemoglobin breaks down, releasing oxygen in solution and this rapidly diffuses into the surrounding tissues

• Haemoglobin has an increasing affinity for oxygen, initial uptake of one oxygen molecule by haemoglobin facilitates the further uptake of oxygen molecules

• Low partial pressure of oxygen corresponds to the situation in the tissue, when partial pressure of oxygen is low, oxygen is released.

• high partial pressure of oxygen corresponds to the situation in the gills, when partial pressure of oxygen is high, oxygen is taken up by haemoglobin

• When oxygen affinity is increased, the dissociation curve is shifted Leftward, and the value is reduced.

• Conversely, with decreased oxygen affinity, the curve is shifted to the right

FACTORS AFFECTING THE OXYGEN-BINDING PROPERTIES OF HB

1. Temp.

2. pᵸ 3. Partial pressure of co₂4. Salt, organic phosphate5. Partial pressure of o₂

TEMP.• Temp.is inversely proportional to Hb saturation.• An increase in temperature will decrease hemoglobin-

oxygen affinity

CO₂ AND pᵸ

• The effect of pH on the blood-O2-binding affinity is describes by the Bohr effect.

• Hemoglobin oxygen affinity is reduced as the acidity increases.

• Active respiration releases CO2 ,increases the partial pressure of CO2.

• Release of CO2 increases acidity i.e. lowers the pH due to formation of hydrogen ions (H+)

• Hydrogen ions bind to hemoglobin decreasing hemoglobin’s affinity for O₂ so O₂ is released from the oxyhemoglobin

ROOT EFFECT

• The Root effect is defined as the oxygen (O2) carrying capacity of hemoglobin reduced at low pH values, even at atmospheric O2 partial pressures (PO2).

• The saturation levels of haemoglobin at this high PO2 are not affected.

• The pH effect- termed Bohr effect, affects O2 affinity.

Hb-oxygen dissociation curve

Hgb Determination

• Cyanmethemoglobin methodThe reagent hemolyzes the erythrocytes which releases the hemoglobin into the solution.

• REACTIVE INGREDIENTS: • -potassium cyanide and potassium ferricyanide

• When blood is mixed with a solution containing potassium ferricyanide and potassium cyanide, the potassium ferricyanide oxidizes iron to form methemoglobin.

• The potassium cyanide then combines with methemoglobin to form cyanmethemoglobin

REFERENCES• Evans David H.,The Physiology Of Fishes.Second

Edition.102.• Anthony P. Farrell., Encyclopedia Of Fish Physiology:From

Genome To Environment. 2,887-895,921-925.• Cox Michael M.and Nelson David L.,Lehninger Principles

Of Biochemistry.Fifth Edition.154-156.• https://en.wikipedia.org/wiki/Hemoglobin.• www.ventworld.com/resources/oxydisso/dissoc.htm.

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