chapter 10

co-operativity and the quaternary shift

• binding of O2 at one site of Hb leads to strain on T state that tears away C-term ion pairs

• Hb takes R state conformation —> increasing empty binding sites affinity for O2

• O2 binding becomes more efficient

changes in ionic interactions during T—> R transition

• t-state

  • formation of ion pairs increases pka values

  • less acidic; keep protons; higher pH

• r-state

  • ion pair interactions are absent, decrease in the pKa values

  • more acidic; will give up protons; lower pH

other effectors for Hb’s affinity for O2

1. [H+] - Bohr effect

2. [CO2]

3. [2,3-Bisphosphoglycerate] [BPG]

Bohr effect

• Christian Bohr observed that the decrease in O2 binding affinity of Hb in response to a decrease in pH

• has important physiological functions in transporting O2 from the lungs to respiring tissue

• related to blood buffering system —> constant pH for physiological rxns

• H2CO3 ⇌ HCO3- + H+ (carbonate acid, bicarbonate)

blood buffering system

• pH decreases:

  • increase in [H+] from metabolic rxns

  • equil. shifts toward H2CO3 (reactant)

• H2CO3 loses H2O to produce CO2, that is expired from lungs

• pH increases:

• decrease in [H+]

• breathing is adjusted so more CO2 in the lungs can be introduced to the blood for conversion to H2CO3

• CO2 in lungs goes to blood —> H2CO3

carbonic anhydrase

• in muscles:

  • respiring tissues product waste CO2

  • Carbonic anhydrase converts this CO2 into H+ and bicarbonate

  • H+ is like a key that locks T-state

  • Hb drops O2 to feed tissues and picks H+

• in lungs:

  • high lvls O2 force Hb to bind O2 switching to R state

  • switching to R state forces Hb to dump H+

  • HCO3- recombines with these H+ protons turning back CO2 gas vi carbonic anhydrase so you can exhale it out

Hb as a CO2 transporter

• carbamation: COO-/NH/ester added to a molecule

• some CO2 transported as HCO3- is carried in blood, some CO2 binds to the N-term amino groups of the Hb subunits to form carbamates

• reaction aids in the transport of CO2 to lungs

• bound carbamates can also form ion pairs - stabilizing deoxy Hb (T-state)

  • Hb is responsible for 50% of CO2 transported by the blood to be expelled by the lungs

T form and CO2

• binds mores CO2 than the R form and because H+ is released upon binding, the R form is shifted to T form and O2 release is favored.

BPG

• BPG binds to T state & stabilizes it

• decreases Hb affinity for O2

• arteries (100 torr), Hb 95% saturated with O2

• veins (30 torr), Hb 55% saturated with O2

  • presence of BPG helps

  • helps Hb “unload” O2 to tissues

Hb binding of BPG

• BPG binding site = T states central cavity

• quaternary shift narrows the central cavity causing Hb to release BPG

• 1 BGP molecule per Hb tetramer

Fetal Hb

• low BPG affinity

• fetus gets O2 from mom’s circulation

• fetal Hb a2g2 (g chain is a variant of b)

• y chain has Ser in place of His 143

Allostery

• a separate control site distinct from the main working site

• binding of a ligand at one site affects the binding of another ligand at another site

• Hb is a allosteric protein

• allosteric effectors: (-) —> diminish Hb’s affinity for O2

  • CO2

  • BPG

• CO2 and BPG bind at sites other than the O2 binding site AND effect Hb’s ability to bind O2

• joseph Barcroft observed that purified Hb binds O2 with higher affinity than Hb in whole blood

model for allostery (symmetry model)

• based on t & r state

• conformational change alters affinity for a ligand

• all subunits either t or r (hybrid states strictly forbidden)

• 2 problems of the model:

  • all t or r NO mixed states

  • only account for (+) cooperativity

Sequential model

• “domino effect” subunits change one by one as O2 binds

• hybrid states allowed

• induce conformational changes in neighboring subunits that influence that subunits affinity for ligand

Allostery of Hb

• O2 binding to Hb involved features of both models

• conf. change in t —> r is concerted (symmetry model)

• ligand binding to the t states does result in small tertiary structural changes that are ultimately required to induce Hb’s quaternary shift (sequential model)

• ligand protein interactions are complex

mutations of Hb

• Hemolytic anemia - deficiency in rbc, compromises O2 delivery to tissues

• cyanosis - O2 binding site mutation in a or b subunit (methemoglobin subunit) leads to oxidation of Fe2+ (Fe3+) and reduces O2 binding cooperativity, bluish skin color results due to increase amount of methemoglobin in blood

• polycythemia - mutations increase Hb’s O2 affinity —> increased rbc’s compensate for less O2 being delivered leading to a ruddy complexion

aa and sickle cell anemia

• single aa substitutions cause sickle cell anemia

• 25% African Americans carry the sickle cell gen (Hb S)

• heterozygous individuals can suffer from mild sickle cell anemia

• homozygous recessive suffer from sickle cell anemia

  • mis shaped rbc can’t pass through capillaries properly

  • can be fatal

sickle cell anemia is a molecular disease

• polymerization of Hb S arises cuz Val replaces His on surface beta chains

• “block” extending from Hb S represents Val side chains

• can insert into hydrophobic pockets in surrounding Hb S molecules

Hb S & Malaria

• Hb S heterozygotes are more resistant to malaria

• malaria is a major cause of death in Africa, so the populations who is heterozygous for Hb S in malarial regions is increased

  • adaptive advantage

• similar to euro descendants and CF

  • those that are heterozygous for CF are more resistant to cholera