MB - Haemoglobin II

Question 1

Why is pure Hb less sigmoidal?

Answer 1

Allosteric regulators bind to Hb and alter affinity for O2

Question 2

What are allosteric regulators and how do they affect hemoglobin?

Answer 2

Allosteric regulators like 2,3-bisphosphoglycerate (2,3-BPG) bind to hemoglobin and alter its affinity for O2

Question 3

How does 2,3-BPG regulate hemoglobin?

Answer 3

2,3-BPG binds to the cavity in hemoglobin, stabilizing the low-affinity T form. It cannot bind to the R form.

Negative charges on BPG interact with positive charges on β subunits

• Impedes subunit movement and favoring the T form

Question 4

What is required to dislodge BPG?

Answer 4

2O2 need to bind to alpha subunits to dislodge BPG and then O2 can bind to beta subunits

Question 5

How do genetic deficiencies related to 2,3-BPG levels affect hemoglobin? (2)

Answer 5

Hexokinase deficiency (↓[2,3-BPG]):

• Less sigmoidal curve, more likely to be in the relaxed R state

Pyruvate kinase deficiency (↑[2,3-BPG]):

• More sigmoidal curve, more likely to be in the tense T state

Question 6

What is the Bohr effect, and how does it regulate hemoglobin?

Answer 6

The Bohr effect is the rightward shift of the hemoglobin saturation curve due to increased H+ and CO2 levels, reducing O2 affinity and promoting O2 release to tissues.

• H+ protonates His146, forming a salt bridge that stabilizes the T form.
• CO2 contributes by increasing H+ levels and forming carbamates that also stabilize the T form

Question 7

How do chloride ions regulate hemoglobin? (2)

Answer 7

Chloride ions enter red blood cells and stabilize the T form in two ways:

• Coating positive residues lining the 2,3-BPG cavity, reducing repulsion.
• Cross-linking Val1α2 and Arg141α1, enhanced by protonation in the Bohr shift

Question 8

What is the importance of C-terminal salt bridges in hemoglobin cooperativity?

Answer 8

Removal of Arg141 or His146 reduces cooperativity (lowers the Hill constant), as these C-terminal salt bridges are essential for the cooperative transition between T and R states

Question 9

How does carbon monoxide (CO) interact with hemoglobin?

Answer 9

CO binds to hemoglobin with x250 affinity than O2, forming carboxyhemoglobin (all-R state).

• At high CO levels, this can be fatal by preventing O2 binding.

Question 10

What acts to weaken the interaction between Haemoglobin and Carbon Monoxide?

Answer 10

Distal His weakens interaction→↓200x

Question 11

How does nitric oxide (NO) interact with hemoglobin?

Answer 11

NO binds to hemoglobin with x10,000 affinity, oxidizing Fe2+ to Fe3+ and forming nitrate.

• Oxyhemoglobin (but not free O2) reacts with NO, displaying nitric oxide dioxygenase activity

Question 12

What is methemoglobin? (4)

Answer 12

• Oxidised form with Fe3+
• Normally <1% of blood Hb
• Brown
• All relaxed form (like myoglobin)

Question 13

What is an example of an inherited deficiency related to Methaemoglobin?

Answer 13

Inherited deficiency of Methaemoglobin reductase

• Methaemoglobinaemia
• Skin turns blue (cyanosis)
• Treated with methylene blue

Question 14

What is Hemoglobin M?

Answer 14

Hemoglobin M is locked in the all-T (low affinity) form due to a single amino acid change that stabilizes the Fe3+ form of the heme.

• An oxygen in a new side chain interacts with the iron

Question 15

What is Hemoglobin S?

Answer 15

• Abnormal HbS distorts RBC
• Only affects deoxy (T) form
• Point mutation in beta subunit
• Glu6→Val6

Charged→Hydrophobic

Hydrophobic interactions between Val and Leu/Phe on another Hb molecule causes aggregation.

Question 16

What is Hemoglobin H?

Answer 16

Thalassaemias are a lack of one or other subunit of Hb

α-thalassaemia – lack of α subunit

• β4 Hb forms (HbH)
• No cooperativity; high affinity
• Poor O2 release to tissues

β-thalassaemia – lack of β subunit

• α subunits aggregate
• Immature RBC destroyed
• Anaemia

Question 17

Why the difference between α-only and β-only in Haemoglobin H?

Answer 17

Two copies of α gene, one copy of β →More α than β made

α normally binds to Alpha Haemoglobin Stabilizing Protein (AHSP) to keep it soluble, before being transferred to β

• In the absence of β [α] > [AHSP] → aggregation of α

Question 18

What is Hemoglobin F?

Answer 18

Fetal haemoglobin

• Start with Zeta instead of alpha
• Start with epsilon (turns into gamma γ) and then beta

γ subunits have His→Ser substitution so weaker attraction for BPG (↑O2 affinity)

• Transfer of O2 from HbA to HbF in placenta