Download Effect of pH on Hemoglobin Oxygen Affinity H + = O 2 Affinity In

Transport of Oxygen
and Carbon Dioxide
Matthew L. Fowler, Ph.D.
Cell Biology and Physiology
Block 4
Reading
• Guyton Chapters 39 and 40
• Important – pp. 490-492
Learning Objectives
•
Compare and contrast myoglobin to hemoglobin and explain how myoglobin can damage the kidney
•
Describe the barriers that oxygen needs to cross to enter tissue and how this occurs
•
Draw and explain the oxygen binding curve for hemoglobin and myoglobin including the hyperbolic curve for
myoglobin, the sigmoidal curve for hemoglobin, the difference in oxygen affinity of the two proteins, and why these
affinities are appropriate to meet oxygen needs in working muscle versus resting muscle.
•
List and explain the factors that modify hemoglobin oxygen binding affinity.
•
List and provide a brief description of the different forms of hemoglobin including Hb F, HbA, thalasemmia,
methemoglobin, carbaminohemoglobin, and sickle cell and detail how these effect hemoglobin and blood flow.
•
Explain how, temperature, pH, CO2 and 2,3 BPG affect oxygen binding to Hb.
•
Define oxygen partial pressure (tension), oxygen content, and percent hemoglobin saturation as they pertain to
blood.
•
Detail how oxygen, bicarbonate and CO2 are transported in the blood and the Bohr and Haldane effect
•
Explain the basis for how carbonic anhydrase effects hemoglobin and pH.
Overview
•
•
•
•
Oxygen is necessary for metabolism
O2 must be captured as a gas, transferred to a liquid
environment for transport.
CO2 from a liquid environment must be transported to the lungs
and returned to the atmosphere as a gas.
Changes required to achieve this:
1.
Pressure
•
2.
Concentration
•
•
3.
Free gases exert partial pressures based on their % composition
Attempt at equilibrium is a driving force
Gases diffuse (smaller gases more easily); down a concentration
gradient
Solubility
•
•
Bound gases no longer exert a pressure
CO2 is much more soluble than O2
Red Blood Cells
• 4-6 x 106 cells/μL
• Hb in cytoplasm
– ~250 million molecules/cell
– Transports O2… and CO2…
sometimes bad things… more later…
• Complex ECM
– Sialic acid residues provide
negative charge
• Prevent sticking and rolleaux (stacking)
• Extracellular branches of
membrane glycolipids define
blood type
RBC Embryology
• First couple weeks
– Yolk sac
• Middle trimester
– Fetal liver (also spleen/lymph
nodes)
• Last month
– Bone marrow
• After puberty only from rib,
sternum, and vertebra
Clinical Correlate: Ectopic production or
RBC’s can occur in renal cell carcinoma
and hepatocellular carcinoma
Transport of Oxygen
Hemoglobin
• O2 and CO2 transport protein
• Blood chemistry affects hemoglobin
oxygen binding properties…key to function.
• Used to transport O2 due to low
solubility
• O2 binds to Hb at high PO2 levels
• Binding – up to 4 O2
Transport of Oxygen
Hemoglobin
Various Subunits During Development
• Always have 2 α-chains
• Fetal Hemoglobin (HbF)
– α2γ2 (primary)
• Post-Birth Hemoglobin
– HbA = α2β2
– HbA2 = α2δ2
Sickle Cell Anemia
Hemoglobin
•
•
•
•
•
•
A single amino acid change (Val6Glu) in the Hb β chain (Hb S)
changes shape of Hb
Causes Hb (S) to polymerize with Hb (A) and form large
insoluble aggregates (clumps)
– Exacerbated by low pH
Insoluble Hb has less affinity for O2
Hb insolubility results in sickling of cells
Sickled cells act as thrombi in capillaries
Result is ischemia manifesting as pain and/or hemolytic crisis
–
 RBC lysis
•  Hb levels
•  Indirect bilirubin levels
•  Reticulocytes
•  LDH
Abnormal States
Methemoglobin (MetHb)
•
•
•
•
•
•
Fe3+ (ferric) state = abnormal (methemoglobin)
Fe2+ (ferrous) = normal hemoglobin
1% in normal person
Methemoglobin cannot carry oxygen
Blood with high concentrations of metHb is bluishchocolate-brown in color
Inorganic nitrites promote ferrous (Fe2+) to ferric (Fe3+)
NADH-dependent methemoglobin reductase
MetHb  Hb
•
Tx. Methylene Blue
Cyanide (CN) Treatment
Methemoglobin (MetHb)
•
Cyanide inhibits cytochrome c
oxidase
–
•
Final step in the electron transport chain
Amyl nitrite used to treat CN poisoning
–
Converts Hb to MetHb
Treatment Mechanism
1.
2.
3.
MetHb binds (CN)
CN-MetHb = Cyanomethemoglobin
(non-toxic)
Cyanomethemoglobin metabolically
eliminated
Carbon monoxide (CO) Toxicity
Carboxyhemoglobin (COHb)
CO has a higher affinity for Hb than O2 or CO2
–
–
Binding of CO to Hb forms carboxyhemoglobin (COHb)
Shifts curve to the left
•
Eventually results in hyperbolic curve
Treatment Mechanism
1.
2.
3.
Remove from CO and supplement O2
Higher O2 than CO allows O2 to out-compete CO for
hemoglobin sites and increases dissociation of CO
HCO3- can be administered if for concurrent metabolic acidosis
Storage of Oxygen
Myoglobin
• Stores oxygen in muscle for emergency use
– Reserve for O2
• Higher in slow twitch muscles
• Pathological - not normally in circulation
(lysis of cells) can lead to kidney damage
• Binding - 1 O2
Rhabdomyolysis
Myoglobin
Breakdown of muscle fibers that Rhabdomyolysis and Renal Failure
from lysed muscle cells circulated in blood (myoglobinuria).
leads to the release of myoglobin 1.2. Myoglobin
Myoglobin interacts with Tamm-Horsfall protein in the nephron .
3. Casts (solid aggregates) obstruct the normal flow of fluid.
into the bloodstream.
4. High levels of uric acid and acidification of the filtrate increase cast
formation.
5.
Under conditions of hypotension, decreased renal perfusion leads to uric
• Results in acute renal failure
acid crystal formation in the renal tubules, resulting in obstruction.
6. Iron released from the heme generates ROS that damage the kidney cells.
• Sensitive marker for muscle
7. These processes lead to acute tubular necrosis, the destruction of the cells
of tubules.
injury
– Statins
– Severe burns
– Myocardial infarction
•
Though a poor indicator for MI
Result
Glomerular filtration rate falls and the kidney is unable to perform
its normal excretory functions.
Outcome
Disruption of electrolyte regulation, hyperkalemia, and interference
with vitamin D metabolism resulting in hypocalcemia.
Hemoglobin has Cooperative
Binding of O2
O2 is never fully unloaded from Hb
Random Sample
•
PO2 of 27 mmHg = 50% O2 Saturation
Normal venous blood
• PO2 of 40 mm Hg = 75% O2 Saturation
Normal Arterial Blood
• PO2 of 100 mm Hg = 96% O2 Saturation
Factors that Modify Hemoglobin
Oxygen Binding Affinity
•
•
•
•
•
Blood pH
CO2 levels
2,3-bisphosphoglycerate
Temperature
Hb chain composition
– HbF has higher affinity for O2
• Allows fetus to sequester more oxygen
from the mother
Blood pH
Normal blood pH
7.35 - 7.45
• < 7.35 = acidosis
• >7.45 = alkalosis
Regulation of Blood pH
Blood pH is regulated using the enzyme
carbonic anhydrase.
In the lungs:
1.
RBC containing H+ bound Hb return to the lungs
2.
On its way it pulls in HCO3- from the blood in exchange
for Cl- using the chloride-bicarbonate antiporter
3.
HCO3- and H+ form H2CO3 (acid)
4.
Carbonic anhydrase converts the acid to CO2 and H2O
5.
CO2 is exhaled from the lungs and water leaves via
osmosis or an aquaporin.
6.
The absence of H+ results in an increase in Hb O2
affinity.
Regulation of Blood pH
Blood pH is regulated using the enzyme
carbonic anhydrase.
In the tissues:
1.
RBC picks up CO2
2.
Carbonic anhydrase combines the CO2 with H2O and
forms H2CO3 which dissociates into HCO3- and H+
3.
HCO3- leaves the RBC in exchange for Cl- using the
chloride-bicarbonate antiporter
4.
The presence of H+ assists in the displacement of O2
from Hb
5.
H+ bound Hb is then returned to the lungs.
Regulation of Blood pH
Important Points
• Blood pH is regulated using the
enzyme carbonic anhydrase
• CO2 diffuses (uncharged)
• HCO3- and Cl- require protein
(charged)
• Most CO2 is transported as HCO3• Things move based on concentration
in an attempt to establish equilibrium.
– Le Chatelier's principle
The Bohr Effect
CO2 acts as a weak acid in the
presence of H2O resulting in the
production of H+ ions shifting the
Hb Saturation Curve to the right.
How? Carbonic anhydrase
Hemoglobin as a Buffer
•
O2 and H+ are competing for the same site on
Hb
•
During exercise you produce lots of CO2
•
CO2+ H2O  HCO3- + H+
•
H+ = O2 Affinity = O2 unloading from Hb
–
•
Affinity – a protein’s ability to capture a substrate
Since Hb can bind H+ it can act as a
physiological buffer
Hemoglobin and CO2
Carbaminohemoglobin
• CO2 affects the blood buffer
system and Hb O2 binding
• CO2 can bind covalently to
the N-termini of Hb chains
• Estimated that 15% of CO2 in
blood is carried by Hb in this
way.
The Haldane Effect
Binding of O2 to Hb reduces Hb
CO2 affinity
• Deoxygenation of the blood
increases its ability to carry carbon
dioxide
• Conversely, oxygenated blood has
a reduced capacity for carbon
dioxide.
Effect of pH on Hemoglobin
Oxygen Affinity
H+ = O2 Affinity
In the muscles/tissues:
1.
Metabolism = H+ (pH)
2.
H+ (pH) = O2 requirements
3.
H+ (pH) = O2 affinity
4.
O2 affinity = O2 binding to Hb
5.
O2 binding to Hb = O2 for tissues
Take Home: Optimized for O2 release
Effect of pH on Hemoglobin
Oxygen Affinity
H+ = O2 Affinity
In the lungs:
1.
Exhalation = CO2
2.
CO2 = H+ (pH)
3.
H+ (pH) = O2 affinity
4.
O2 affinity = O2 binding to Hb
Take Home: Optimized for O2 binding
Effect of pH on Myoglobin
Oxygen Affinity
No Effect!!!
•
•
Myoglobin follows hyperbolic
saturation
Not a cooperative-binding system
Effect of 2,3-BPG on
Hemoglobin Oxygen Affinity
•
2,3-bisphosphoglycerate (2,3-BPG)
binds allosterically with greater affinity
to deoxygenated Hb than oxygenated Hb
–
aka. Allosteric effector
•
•
•
Interacts with β-subunits
Decreases Hb O2 affinity
Promotes unloading of O2 at tissues
•
HbF does not recognize 2,3-BPG
–
Allows for HbF to more readily bind O2 from the
mother for fetal use
Effect of 2,3-BPG on
Hemoglobin Oxygen Affinity
• Whole blood consists of Hb in the
presence of both CO2 and 2,3-BPG
•
In the absence of 2,3-BPG, oxygen binding to Hb follows a
rectangular hyperbola
•
The sigmoid binding curve is only observed in the
presence of 2,3-BPG
Summary
•
•
•
•
•
Hemoglobin has a sigmoid O2 binding
curve
Binding of O2 to Hb can be altered by
different factors
Hb can be altered by genetics (e.g. sickle
cell)
Oxygen is transported bound and unbound
in the body and is dependent on partial
pressures
Total oxygen in the body is a sum of bound
and unbound