Fish hemoglobin is similar to other vertebrate hemoglobin but has some key differences. It is a tetrameric molecule composed of two alpha and two beta globin chains, each containing a heme group. Unlike other vertebrates, fish hemoglobin has fewer histidine residues per globin chain and typically has serine instead of cysteine at position 93 of the beta chain. Fish hemoglobin also exists in multiple forms that differ in oxygen affinity and other properties. The heme group allows hemoglobin to reversibly bind oxygen, with factors like temperature, pH, salt concentrations, and partial pressures of oxygen and carbon dioxide impacting binding. Hemoglobin transports oxygen from gills to tissues via changes between tense and relaxed conformational states.
2. 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
3. STRUCTURE OF HEMOGLOBIN
• hemoglobin -most fishes is a tetrameric molecule
• molecular weight- 60 000–70 000Da.
• consists of four globin chains- two α- and two β chains.
• 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.
5. • 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
6. 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.
9. 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.
10.
11. Hb DIFFERENTION IN FISHES
Polymorphism 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;
Monomeric
Tetrameric
12. 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.
13. 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%).
14. 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.
17. • 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.
18. 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
19. • 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
20.
21. FACTORS AFFECTING THE
OXYGEN-BINDING PROPERTIES
OF HB
1. Temp.
2. pᵸ
3. Partial pressure of co₂
4. Salt, organic phosphate
5. Partial pressure of o₂
22. TEMP.
• Temp.is inversely proportional to Hb saturation.
• An increase in temperature will decrease hemoglobin-
oxygen affinity
23. 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
24.
25. 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.
28. Hgb Determination
• Cyanmethemoglobin method
The reagent hemolyzes the erythrocytes which
releases the hemoglobin into the solution.
• REACTIVE INGREDIENTS:
• -potassium cyanide and potassium ferricyanide
29. • 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
30. 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.
