P: Haemoglobin & myoglobin

Question 1

Globular haemeproteins:

Answer 1

group of specialised proteins that contain haeme as a tightly bound prosthetic group.

Question 2

Haeme group functions:
Haemoglobin + myoglobin
Soluble guanylyl cyclase
Catalase
Cytochrome

Answer 2

Haemoglobin + myoglobin: oxygen binding
Soluble guanylyl cyclase: binding of vasodilator - nitric oxide
Catalase: binding + decomposition of hydrogen peroxide
Cytochrome: electron binding in electron transport chain.

Question 3

Haeme

Answer 3

Complex of protoporphyrin IX and ferrous iron (Fe2+)
In myoglobin & haemoglobin, Fe2+ in porphyrin ring forms two additional bonds with histidine in globin protein + oxygen.

Question 4

Myoglobin

Answer 4

monomeric haemeprotein located within skeletal, cardiac + smooth muscle cells.

Function:
- Reservoir of O2 within muscle cells to drive muscle contraction during arterial hypoxaemia
- Haeme group an also scavenge excess reactive oxygen species that can damage cells.

Question 5

Structure of myoglobin

Answer 5

• 80% arranged in 8 alpha-helices A-H
• Polar + charged residues on exterior
• Nonpolar, hydrophobic residues in interior
• Tethered into hydrophobic cleft in protein formed by E+F helices which each contain histidine residue:
  1. Proximal histidine (F8) binds Fe2+ in haeme
  2. Distal histidine E7 helps stabilize ferrous form of iron, allowing reversible binding of oxygen to ferrous ion.

Question 6

Haemoglobin

Answer 6

Only found in RBCs, transports oxygen and CO2 in circulatory system.

Haemoglobin structure:
- Four protein subunits with associated haeme group - each structurally similar to myoglobin
- Two alpha and two beta subunits
- One molecule of haemoglobin can transport 4 molecules of oxygen.

Question 7

Deoxyhaemoglobin structure

Answer 7

• Haeme group is nonplanar
• Fe2+ is pulled out of the plane of porphyrin towards histidine residue
• Fe2+ lies approximately 0.4Å outside porphyrin plane

Question 8

Oxyhaemoglobin structure

Answer 8

• Fe2+ is pulled into plane of porphyrin ring.
• Haeme group is planar.

Question 9

T form deoxyhaemoglobin

Answer 9

• Hydrophobic + ionic bonds contstrain movement of four subunits
• Low affinity form

Question 10

R form oxyhaemoglobin

Answer 10

• Fe2+ found to F8 histidine on protein subunit
• Movement of Fe2+ into plane of haeme group upon O2 binding causes changes in quaternary structure of haemoglobin
• Breaking of hydrophobic/ ionic bonds
• Subunits now have more movement
• High oxygen affinity

Question 11

Oxygen binding to haemoglobin

Answer 11

T-R conformational changed caused by oxygen binding to one subunit is transmitted to other 3 monomers in tetramer.

Binding of oxygen to one subunit induces increased binding to the other three subunits.

Question 12

Pulmonary capillaries

Answer 12

• pO2 = 100mmHg
• Cooperativity facilitates rapid binding to HgB
• T-R transitions increase affinity of other HgB subunits for O2
• Saturation = 100%

Question 13

Systemic capillaries

Answer 13

• pO2 = 40mmHg
• On average, one O2 is released from each HgB molecule
• Saturation = 75%
• Sufficient for oxygenation of tissues under resting conditions
• Built-in reserve capacity.

Question 14

Tissue hypoxia

Answer 14

Tissue hypoxia pO2 < 40mmHg
- More O2 is rapidly released from HgB delivering more O2 to tissues
- R-T transitions reduce affinity of other Hb subunits for O2
- Reduced affinity, more O2 unloaded from subunits.
- O2 unloading from Hb makes it “easier” for O2 to be released from other subunits

Question 15

Sigmoidal nature of oxyhaemoglobin dissociation curve:

Answer 15

• Allows HgB to act as transporter of oxygen
• High affinity for O2 in lungs and reduced affinity in tissues

Question 16

Hyperbolic nature of myoglobin dissociation curve:

Answer 16

• Allows myoglobin to act as an O2 reservoir within muscle cells.
• High affinity for O2 at normal muscle pO2 levels
• Rapid release of O2 only at very low muscle pO2 levels.

Question 17

Factors that modulate affinity of haemoglobin for oxygen:

Answer 17

• pH of blood
• Carbon dioxide
• 2,3 diphosphoglycerate: a metabolite of glycolysis pathway.

Question 18

Increased metabolic activity increases:

Answer 18

• Directly interacts + causes structural change in haemoglobin
• Reduces affinity of haemoglobin for oxygen
• Increased unloading of oxygen into tissues
• Alleviates hypoxia caused by increased metabolic activity

Question 19

p50

Answer 19

pO2 at which Hb is 50% saturated.

P50 increases –> higher pO2 required for 50% saturation –> more O2 unloaded for Hb

P50 decreases –> lower pO2 required for 50% saturation –> less O2 unloaded from Hb.

Question 20

Bohr effect

Answer 20

effect of acidity + carbon dioxide on oxyhaemoglobin dissociation curve.

Increased metabolic activity: increased lactic acid production & increased carbon dioxide production

Question 21

Bohr effect: reduction in pH

Answer 21

Causes protonation of histidine residues on Hb.
This allows additional ionic bonds to form between Hb subunits
- Stabilizes T form of Hb
- Lower affinity and increased released of O2
Co2 can also bind directly to free amino groups on Hb
- Forms negatively charged carbamate groups
- More ionic bonds between Hb subunits stabilizing T form.

Question 22

Effect of 2,3 diphosphoglycerate:

Answer 22

• Glycolytic pathway is the only source of ATP in hypoxic tissues
• 2,3 DPG binds to pocket of positively charged amino acids formed by beta-subunits of Hb: this stabilizes T form of Hgb, lowering affinity and increased release of O2
• High [O2] in lungs expels 2,3-DPG –> t-R transition.

Question 23

Carbon monoxide + haemoglobin

Answer 23

Carbon monoxide displaces oxygen, forming carbon monoxyhaemoglobin.

At lower concentrations, binding of CO shifts conformation of other subunits to R form: subunits have higher affinity for O2, so less oxygen released to tissues.

Question 24

Forms of haemoglobin

Answer 24

1. Haemoglobin A (HbA): major form in adults (90%)
2. Haemoglobin A2 (HbA2): minor adult form (2-5%
3. Haemoglobin Gower 1: produced during early embryonic development
4. Haemoglobin F (HbF): synthesised during foetal development.

Question 25

Foetal haemoglobin - affinity to oxygen and why?

Answer 25

• HbF has higher affinity for O2 compared to HbA
• This is due to weak binding of 2,3-DPG to HbF as subunits lack some positively charged amino acids required to bind 2,3-DPG, which promotes T-R transition
• Higher O2 affinity of HbF facilitates transfer of O2 across placenta from maternal red blood cells.

Question 26

Haemoglobinopathies

Answer 26

Family of genetic diseases that result in the production of:
1. Insufficient quantities of haemoglobin molecules
2. Structurally abnormal/defective haemoglobin molecules.
Results in anaemia.

Question 27

Causes of sickle cell anaemia

Answer 27

• Point mutation in B-globin gene: GAG–> GTG, so VAL is encoded instead of GLU
• In deoxy-HgS: Val6 interacts with Phe85 and Val88 of B-chain, causing aggregation of HbS molecules.
• In oxy-HgS: In R conformation, Phe85 and Val 88 are buried inside molecule, so no interaction or clumping.

Question 28

Sickle cell anaemia consequences

Answer 28

• Lower pO2 levels in systemic capillaries: aggregation of HgS –> red blood cells deform to sickle-shape –> obstruction of capillaries + restriction of organ blood flow –> haemolysis
• Anaemia
• Pain + fever
• Organ damage
• Severe infections

Question 29

Thalassaemia

Answer 29

No production/ reduced production of globin subunits. Caused by gene deletions or mutations that block/reduce transcription or translation.

Question 30

B- thalassaemia

Answer 30

• One copy on chromosome 11
• Heterozygotes: B thalassaemia trait
• Homozygous: unpaired alpha subunits cause death of progenitor RBC –> ineffective erythropoiesis + severe anaemia

Question 31

A-thalassaemia

Answer 31

• Two copies of a-genes on each chromosome 16
• Severity depends on number of defective genes: asymptomatic - severe haemolytic anaemic -foetal death).