OXYGEN TRANSPORT AND STORAGE

Myoglobin
• Globular protein, 3° folding, 8 right handed α helices (A – H) with 7-20 AAs each
• Heme – prosthetic group @ hydrophobic crevice; porphyrin ring + central Fe
• Nomeclature – letter of helix + AA position; order of AA residue
• 5 non-helical segments – AB, CD, EF, FG, GH
• 2 non-helical terminal segments – NA1 and NA2, HC1-HC5

Hemoglobin
• Quaternary structure
• Tetramer = 2α and 2β subunits facing each other across a central cavity
• Packing contact @ α1β1 & α2β2, fixed
• Sliding contact @ α1β2 & α2β1, not fixed
• 4 heme groups = 4 oxygen binding sites
• O2 binding @ 6th coordination position of heme
• Val E11 pushes and destabilizes nonperpendicular molecules @ binding plane (i.e. CO) allowing reversible binding
• Conserved AA:
o His F8 (proximal His / residue 93) - always bound to Fe
o His E7 (distal His / residue 64) - binds to O2, then O2 binds to Fe
o PheCD1, Leu F4 – heme contact
o Gly B6 – allows close approach of helices B and E via B6&E8

Type Subunits F2 F3 Info

HbA α2β2 Ala Thr
HbA2 α2δ2 Ser Gln 2%
HbF α2γ2 Ala Gln
Embryonic ζ2ε2 (Gower1), α2ε2 (Gower2), ζ2γ2 (Portland)

Heme
• Prosthetic group in hydrophobic pocket for heme sequestration and prevention of Fe2+ oxidation to Fe3+
• Protoporphyrin ring – organic, 4 pyrroles via methenyl bridges
• Fe2+ atom - @ center or porphyrin ring
• 6 ligands via coordinate bonds
o Four are 4N of porphyrin ring
o 5th – HisF8 or residue 98 or proximal His
o 6th – reserved for oxygen during oxygenation

Oxygenation and Deoxygenation

• Reversible binding of O2 to heme
• Y = pO2 / (pO2 + Kdiss)
o Y = fractional saturation of myoglobin
o Kdiss = dissociation constant
• @ Y = 0.5, Kdiss = Po2 = P50
• Oxygen Binding Curve – reflects oxygen affinity, shows P50
o Positive Cooperativity – binding of O2 to one heme facilitates binding of additional O2
 Y / (1-Y) = pO2 / P50  log Y / (1-Y) = log pO2 / P50  Hill Plot = log pO2 vs log Y / (1-Y)

OBC Shape P50 Cooperativity

Myoglobin Hyperbolic 4 mmHg N/A (nH = 1)
Hemoglobin Sigmoidal 26 mm Hg + (nH < 1)

Hemoglobin Conformations
• T / taut = lower O2 affinity = deoxyhb = dome shaped
• R / relaxed = higher O2 affinity = oxyhb
• Transition from T to R
o 15° rotation of α2β2 relative to α1β1
o Breaking of 8 salt bridges that stabilize T state
o New H bonds @ α1β2 interface = AspG1-AsnG
o Molecular rearrangments – O2 pulls heme Fe  straightening of dome
• molecular strain @ HisF8 from 8° to 0°, Val F65 pushed away

Factors affecting Oxygen Binding

• Temperature - ↑ = OBC right shift
• pH = ↓ = right shift
 o Bohr effect = Hb response to any pH change; no effect on myoglobin
o ↓ pH = His 146 protonated = salt bridge with Asp 94
• pCO2
o carbonic anhydrase  HCO3 + H+  Bohr effect
o carbamination = formation of carbaminohemoglobin + H+
• 2,3 DPG - @ central cavity of deoxyHb, produced by Rapaport-Leubering Shunt
o Central cavity – positive charges (Lys 82, His 2, His 143, NH2 terminals of β chains)
o Lowers O2 affinity by stabilizing T state
o γ subunits of fetal Hb (HbF) = ↓ affinity for 2,3 DPG = leftward shift = ↑ O2 affinity relative to maternal
o Factors:
• ↑ 2,3 DPG – chronic hypoxemia, anemia
• ↓ 2,3 DPG – acidosis, RBC age, stored blood

Carbon Monoxide
• Forms carboxyhemoglobin, by product of heme degradation, 1% of O2 binding sites
• Heme affinity = 25000x than O2
• Can interfere with O2 acquisition by heme
• Shifts OBC left
• HisE7 reduces affinity of linear CO binding to heme
• Optimal binding orientation= 121°

Methemoglobinemia
• Hemoglobin Fe2+ oxidized to Fe3+
• Hb cannot carry O2
• Results when metHb > 1.5%
• Leftward shift

Hemoglobinopathies
• Inherited, substitution or absence of AA in globin chain
• Sickle Cell Anemia – point mutation @ β chain, Glu6  Val = HbS
o Val6 on β chain produces sticky patch at exterior
o Complimentary site @ deoxy HbA or HbS + sticky patch = aggregation = RBC distortion, HbS stabilization
o ↓ pO2 = deoxyHbS aggregation = sickling
o Clinical symptoms: hemolytic anemia, infarctive crisis via occlusion, P50=31 (rightward shift = ↑ 2,3 DPG)
o Oxygenation = ↑ oxyHbS = ↓ deoxyHbS = mask complimentary site = ↓ sickling
o Deoxy HbA from transfusion = no sticky patch

Thalassemia Hb Info
defective gene expression of α globin
α-Thalassemia 2 Silent carrier, one gene deletion
α α-Thalassemia Trait Hb Barts (γ4) 2 gene deletion
Hemoglobin H Hb H (β4) 3 gene deletion
Hydrops Fetalis No α chains
Minor β/β+ to β/β° Mild hypochromic microcytic anemia, asymptomatic
β Intermedia β°/β+ to β+/β+ depends on β+ severity
Major β°/β+ to β°/β° severe anemia, multiple organ damage, transfusion-dependent

CARBON DIOXIDE TRANSPORT AND CHEMISTRY OF RESPIRATION

Carbon Dioxide
• Waste product from metabolism of sugars
• Transported by circulatory system: brought to alveoli  expelled
Respiration O2 Direction CO2 Direction
External Alveoli  capillaries Capillaries  alveoli
Internal Capillaries  cells Cells  capillaries

Transport
• Same as O2 transport:
• Convection transport = tissue  lungs
• Diffusion from ↑ pCO2 (venous blood)  ↓ pCO2 (lungs)
• Differential solubility of O2 and CO2
• Forms in Blood:
Form Amount Info
Dissolved CO2 9-10% (3°) CO2 = 20x more soluble than O2
75-78% (1°) Carbonic anhydrase reaction: CO2 enters RBC  + H2O + carbonic
HCO3-
anhydrase = H2CO3  spontaneously dissociates into HCO3- + H+
 Carbaminohemoglobin 13-20% (2°) CO2 + amino group of hemoglobin

Carbaminohemoglobin Formation
• CO2 + terminal NH2 of aliphatic CHONs
• RBC pH = 7.2 = NH2  NH3
• Removal of some uncharged NH2 via carbamino formation  generate more NH2 + H+
• Hemoglobin is most important:
o More Hb than CHON
o Tetramer = 4 terminal NH2
o DeoxyHb (venous blood) forms carbamino more readily than oxyHb  oxygenation = CO2 release

Regulation
• Buffering – HCO3- and H2CO3 buffering in ionizable groups (i.e. 4 N-terminal NH2 groups, imidazole of His38, phosphate groups)
• Isohydric Mechanism – Hgb can take up H+ from CO2 without pH change

Haldane Effect
• H+ affinity: deoxyHb > oxyHb
• O2 + Hb = ↓ CO2 affinity (CO2 dissociation curve rightward shift, Haldane effect)
• CO2 + Hb = ↓ O2 affinity (Bohr effect)

Chloride Shift
• Exchange of HCO3 and CL between plasma and RBC = maintain electrical neutrality
• cross membrane via Band 3