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1. Oxyhaemoglobin

Combination with Oxygen (Oxy-Hb): The physiological importance lies in the fact that Hb can readily combine with O2. The combination is loose and reversible. The gas is taken up readily at high partial pressures (e.g. in the lungs) and is released as readily at low O2 pressures (e.g. in tissues), thus providing an effective system for transport of O2 from the

atmosphere to the cells of the body. At O2 tensions of 100 mmHg or more, Hb is virtually 100 per cent saturated, approx. 1.34 ml of O2 is then combined with each gram of Hb.

For an average Hb concentration of 14.5 gms%, total O2 which would be carried as oxy-Hb would be 14.5 × 1.34 = 19.45 ml%. To this an amount of 0.393 ml of physically dissolved O2 is to be added taking it to 20 volume%.

Deoxygenated Hb can loosely combine with O2 forming Oxy-Hb, the attachment with O2 occurs with Fe in the haem portion. Fe remains in the ferrous state both in deoxygenated Hb and oxy-Hb. O2 remains attached with the unpaired electrons of Fe.

Each haem can bind only one mol. of O2. Since each molecule of Hb contains 4 mols. of haem; hence one mol. of Hb can maximally combine with four mols of O2.

Factors:The combination is loose and reversible and governed by the following factors:

• Partial pressure of O2 (Po2) favours oxygenation • Partial pressure of CO2 (Pco2) favours dissociation • pH of the medium-acidosis favours liberations of O2.

Haem-haem Interactions: Combination of O2 with one haem situated on α-chain, which can permit a molecule of O2 to enter, brings about conformational changes, so that O2 can enter into other haem groups situated on β-chains, the valine residue is removed permitting O2 to enter. The increase in O2 affinity by haem group on

β-subunit, after combination of O2 with α-subunit is called “haem-haem interactions”.

Haem-linked groups: The “affinity” of Hb for O2 decreases with decrease in pH, in the physiological range. The converse effect occurs with increase in pH. On the other hand, oxygenation of Hb results in liberation of H+ _ions into the medium. Thus oxy-Hb is a stronger acid than deoxygenated Hb. The interrelation of oxygenation and ionization suggests the presence in Hb of ionizable group located in sufficient proximity to the haem ring to be influenced by the state of oxygenation. These have been identified as “imidazole” rings of histidine residues, No- 87 on ααααα-subunit and No-92 on βββββ-subunit.

Two states of Hb: Haemoglobin can occur in two states:

(i) ‘R’ state: This is a relaxed state and it is oxygenated Hb.

(ii)‘T’ state: This is a tense/taut state and it is de- oxygenated Hb.

These two forms are interconvertible and each form has its own equilibrium constant (KR & KT) for the binding of O2. De-oxygenated ‘T’ form is stabilised by salt bridges and BPG in a central pocket.

Salt Bridges

These are defined as ionic bonds between +vely charged nitrogen atoms and –vely charged O2 atoms. On

oxygenation of ‘haem’ of α-subunit which can easily admit one mole of O2, the following conformational changes take place in the Hb molecule (“T” form → → → → ‘R’ form):→

• Rotation of one pair of rigid subunits α2 β2 through 15o _{along the long axis, to the other rigid pair of subunit} α1 β1 occurs.

• As a result of rotation, salt bridges are broken. • BPG cannot be held in central pocket as it cannot form

salt bridges.

• Valine residue in haem pocket of β-chain is removed and this can now admit a molecule of O2(Table 11.2). Table 11.2: Differentiation of ‘T’ form and ‘R’ form

‘T’ form (Taut form) or (Tense form) ‘R’ form (Relaxed form)

1. Deoxygenated Hb 1. Oxygenated Hb

2. The rigid subunit α1β1 and α2β2 are close to the long axis 2. Rotation of 15o _{along the axis}

3. Salt bridges are numerous and plenty and intact 3. Due to rotation the salt bridges are broken

4. Valine residue covers the haem pocket of β-subunit and does 4. Valine residue in haem pocket of β-subunit is removed and

not allow entry of O2 O2 can enter

5. BPG can bind and helps in retaining the salt bridges 5. BPG cannot bind as the salt bridges are broken

6. Fe++ _{is 0.07 nm out of the plane of porphyrin ring 6. Fe}++ _{comes in plane of porphyrin ring}

7. Affinity of O2 is low 7. Affinity to O2 becomes high (Haem-haem interaction)

SECTION TWO

Absorption spectrum of Oxy-Hb: On spectroscopy, oxy- Hb shows two characteristic bands.

• ααααα-band: Narrow-band in yellow portion of the spectrum at 597 mμ nearer to D-Line

• βββββ-band: Wider broad-band, nearer to E-Line in green part of spectrum at 542 mμ.

2. Carboxy-Hb: Combination with CO

CO combines with haem portion of Hb to form carbon monoxide Hb (also called as carboxy-Hb or carbonyl Hb).

Characteristics

• It is a much firmer combination, as compared to oxy-Hb and not reversible

• Affinity of Hb to CO is 210 times more than O2 • Lethal action is due to inhibition of cytochrome

oxidase of electron transport chain and thus stops cellular respiration.

Poisoning by CO is a common danger of modern life. Carbon monoxide is particularly dangerous as:

• It is colourless and odourless and hence cannot readily be detected if present in atmosphere, and

• Its action is insidious and rapid. The victim frequently becomes unconscious in a few minutes and death often follows quickly.

Causes

• Incomplete combustion of carbonaceous materials • Automobile exhaust gas (4–7%)

• Chimney gases and smoke

• Found as a constituent of illuminating gas (derived from coal or oil) in which its presence varies from 4 to 40 per cent.

An individual inhaling CO from above mentioned sources can become a victim.

Clinical features: Depends on concentration of carboxy- Hb in blood (% of Hb saturated with CO).

Toxic Effects of Carboxy-Hb

% of Hb saturated Symptoms

with CO

0 to 10% None

10 to 30% Possibly slight headache, throbbing in temples.

30 to 50% Severe headache, weakness and dizziness, nausea and vomiting, dim vision and possibly collapse. 50 to 60% Above + unconsciousness, coma with

intermittent convulsions, Cheyne- Stokes respiration.

60 to 80% Above + depressed heart action and respiration, respiratory failure and death.

Tests to Identify Carboxy-Hb

• Physical examination of blood: It shows a cherry-red colour.

• Chemical test:Dilution test:To dilute the suspected blood, after treating it with a little NaOH and compare the colour with normal blood similarly treated. Normal blood shows a greenish hue after such treatment whereas blood containing carboxy-Hb remains pink.

• Absorption spectra: These resemble like oxy-Hb, but the two bands are slightly nearer to violet end of the spectrum. On treatment of blood sample with sodium hydrosulphite, there is no change of absorption bands (two bands persist) in case of carboxy-Hb while the two bands of oxy-Hb are changed to one broad β-band (α-band disappears) as Oxy-Hb is reduced to deoxy- Hb.

Figure 11.3 gives absorption spectra of different derivatives.

Fig. 11.3: Absorption spectra of different HB-derivatives

SECTION TWO