F8i / 23A13 / 20A11: Describe the structure and function of adult haemoglobin

23A13: Exam Report

Describe the structure and function of adult hemoglobin

25% of candidates passed this question.

Good answers provided detail about the specific composition of haemoglobin and related the pertinent features of the molecule to its functions in the carriage of oxygen, carbon dioxide and its role in acid-base balance, including the appropriate mechanistic description.

Many candidates provided information on the production and breakdown of haemoglobin which was not required. Many candidates provided an unnecessary amount of detail and diagrams of the oxygen haemoglobin dissociation curve which did not score additional marks.

20A11: Exam Report

Describe the structure and function of adult haemoglobin.

57% of candidates passed this question.

Marks were awarded for the two components of this question – structure and function. The structure component was often only briefly described with a cursory overview provided; however, this component contributed around half of the available marks. Many candidates were unable to accurately describe the structural components of the haemoglobin molecule. The functional component was handled better – however much time was wasted with detailed drawings of the oxyhemoglobin curve (not many marks awarded for this). The basic function of haemoglobin carriage of oxygen and carbon dioxide was known, but detail was often missing about its role as a buffer or its role in the metabolism of nitric oxide.

F8i / 23A13 / 20A11: Describe the structure and function of adult haemoglobin

Definition

A molecule of 4 protein chains, each carrying a heme group: MV 65,000 Daltons

Each RBC consists of 200 – 300 million molecules of Hb

Structure

2 parts: heme + globin
Each heme moiety consists of PROTOPORPHYRIN RING & central iron ion in ferrous state (Fe²⁺)
Heme synthesis occurs in RBC cytosol & mitochondria
Fe²⁺ forms 6 bonds with heme moiety; 5 bind Fe²⁺ firmly, 1 binds O₂
4 polypeptide chains determine type of Hb
- HbA = 2α + 2β globin chains
- HbF = 2α + 2γ globin chains
- HbA2 = 2α + 2δ globin chains
Altered polypeptide chains create variants important to know because alter body’s capacity to transport O₂

NB: 98% of normal adult Hb is HbA

Function

Transport O₂ from lungs → tissues
Transport CO₂ from tissues → lungs as CARBAMINO Hb
Buffer H⁺ formed in RBC
NO metabolism

1. Transport O2 from Lungs → Tissues

O₂ binds reversibly to heme
4 hemes → each Hb can carry 4 x O₂
Hb is an allosteric protein
The binding of O₂ to one heme group ↑affinity of remaining heme groups for O₂
This +ve cooperativity means that DeoxyHb & oxyHb have very different structures
This shape is maintained by electrostatic bonds b/w α-acid sequences

DeoxyHb: electrostatic bonds are strong, hiding heme deep in crevice, making it difficult for O₂ to access heme this quaternary structure → _KATENSE FORM

OxyHb = binding of first O₂ to a heme changes the electrostatic bonds. Relaxes the crevice holding heme and enlarges the access → transmits this to other globin chains by distracting their electrostatic bonds & the quaternary structure is altered
- ↑ other globin affinity for O₂
- _KA RELAXED FORM when 4O₂ bound to 1Hb

Huffner Constant
- O₂ binding capacity of Hb = amount of O₂ in mL each Hb can carry
- _KA HUFFNER’S CONSTANT = 1.3mL per 1g of Hb
The conformational state of Hb (R/T) is also altered by other factors, which influence strength of electrostatic bond:
- CO₂
- pH
- Temperature

The Oxyhaemoglobin Dissociation Curve
- O₂ binds reversible with ferrous iron of Hb to form oxyhaemoglobin

Hb + O₂ ⮂ HbO₂

Forward reaction occurs in lungs due to ↑PO₂
Backward reaction occurs in tissues due to ↓PO₂
Curve is SIGMOID SHAPE due to COOPERATIVITY
Affinity for O₂ is lowest when first molecule binds to ‘TENSE’ deoxygenated Hb
As each subsequent molecule binds, the gradient of the curve increases
As PO₂ increases, all the Hb – O₂ binding sites become occupied & the curve levels off
3 important points on ODC:
1. Arterial PO₂ where Hb 100% saturated (PO₂ 100 = SpO₂ 97%)
2. Venous saturation (PO₂ 40 = SpO₂ 75%)
3. P₅₀ (P₅₀ = 27mmHg PO₂ = SpO₂ 50%)

P₅₀ = the partial pressure of oxygen in blood where Hb is 50% saturated
P₅₀ is a measure of oxygen affinity
∴It is useful to compare changes in the position of the ODC
P₅₀ for any single form of Hb is variable → the hydrogen bonds & ionic interactions within Hb result in altered affinity of Hb for O₂

R) Shift

↑2,3 DPG

↑Temp

↓pH

↑CO₂

L) Shift

↓2,3 DPG

↓Temp

↑pH

↓CO₂

An ↑[H⁺] will ↓Hb affinity for O₂
DeoxyHb binds more actively with H⁺ ions
∴as pH ↓so does Hb affinity for O₂

_KA BOHR EFFECT: ↓ O₁ affinity at low pH & ↑ O₂ affinity at high pH

The Bohr Effect is important because it offloads O₂ to metabolically challenged areas
CO₂ has a similar effect because results in ↑[H⁺]

CO₂ + H₂O ⮂ H₂CO₃ ⮂ H⁺ + HCO₃^–

2, 3 DPG

2,3 DPG is a highly anionic organic phosphate
Produced by Rapport – Luebering Shunt

This shunt comes off the glycolysis pathway
Level of 2,3 DPG determined by balance of synthesis/degradation
Activity of DPG Mutase ↑ with ↑H⁺

∴↓Hb affinity for oxygen & displacing ODC to RIGHT
2,3 DPG is ↑in ANAEMIA & HIGH ALTITUDE
∴tissue hypoxia of anaemia partially corrected by ↑P₅₀
The ↑RESP ALKALOSIS at altitude has much more pronounced effect than the ↑2,3 DPG & at high altitude ODC shifts LEFT

2. Transport CO2 from Tissues → Lungs as CARBAMINO Hb

6% of CO2 in blood is as Carbamino Compounds
Carbamino compounds are formed when CO2 binds to Hb
Side chain of valine residues have an N-terminal amino group to which the CO2 will bind to form carbamates
The carbamate stabilizes the Hb in the Tense Form
This lowers to affinity of Hb for O2 (KA Haldane Effect)
Carbamino formation accounts for 2/3 of the Haldane Effect

3. Buffer H+ formed in RBC

Buffer = a substance with the capacity to bind/release H⁺ to minimise pH ∆
Hb is an Intracellular buffer because exists inside RBC but classified as an extracellular buffer because buffers extracellular acids rapidly
Exists in RBC as weak acid (HHb) & its potassium salt (KHb)
Buffers via the imidazole residues of histidine residues with pKa 6.8
Each Hb has 38 histidine residues – large buffering capacity
Hb is present in high concentrations → 15g/dL
DeoxyHb kPa = 7.9
OxyHb kPa = 6.8
When Hb is deoxygenated → conformational ∆ alters pKa of histidine residue & makes it more effective buffer at physiological pH → this makes up the other 1/3 of the HALDANE EFFECT (deoxygenated blood can carry more CO₂)
Hb will also buffer by binding CO₂ to form carbamino compounds

Clinical Importance

Buffers H⁺ & CO₂ from cellular metabolism
Buffers fixed acids prior to renal excretion
Assists CO₂ carriage in blood
- Conformational ∆ to deoxyHb
- 7 mmol H⁺ can be taken up
- ∴7 mmol CO₂ enters venous blood with minimal pH Δ

Reactions in RBC

Cell metabolism produces CO₂
CO₂ diffuses into RBC
CO₂ + H₂O ⮂ (Ca) H₂CO₃ ⮂ H⁺ + HCO₃^–
H⁺ taken up by histidine residues → buffered
HCO₃^– exchanged with Cl^–via antiporter → HAMBURG EFFECT
Some CO₂ binds terminal amino groups → CARBAMINO COMPOUNDS

4. No metabolism

NO is produced by endothelial cells by NO synthase
NO is highly vasoactive:
- Vasodilates smooth muscle
- Inhibits platelet activation
- Induces expression of endothelial adhesion molecule
Its rapid reaction with Hb is the major mechanism for disarming its bioactivity:
- HbO2 + NO → MetHb + NO3^–

This forms methemoglobin (in which heme irons are ferric) and nitrate

Preservation of NO activity by Hb occurs when deoxygenated hemes form iron-nitrosyl Hb:
- Hb + NO → HbNO

This reaction can occur at any of the 4 hemes when they are deoxygenated

The rate of this reaction is extremely slow, so this ‘capturing’ of NO is not an effective way to transduce NO bioactivity