Course:SOMBlock3IQ1/Week 1/Case 3 Hemoglobin
Goal: Students will understand the unique characteristics of the globin family of proteins (myoglobin and the hemoglobins) and their role in oxygen transport.
[edit] Learning Objectives
1. Describe the structure of hemoglobin A and myoglobin.
- Hb A – tetramer: 2 a subunits, 2 B subunits; function as pair of identical aB dimmers; subunits held together by H-bonds, ionic bonds
- Mb – globular protein; 1 polypeptide
- Rich is a helices: 8 right-handed
- Polar: w/ 2 exceptions, interior = hydrophobic
- Both contain heme
2. Describe how oxygen binds to hemoglobin A and myoglobin.
- Oxygen binds cooperatively to Hg A: one O2 binds and increases O2 binding affinity to other subunits – described by Hill coefficient – displays sigmoidal behavior – releases O2 at lower pO2 than Mg
- Oxygen binds noncooperatively to Mg: described by Michaelis-Menton equation – form of oxygen storage; O2 only released at pO2 <5 mm Hg
3. Compare and contrast the binding of oxygen to hemoglobin A and myoglobin and explain the significance of this difference.
- Hg A: 4 O2 binds to Hg A at high pO2 and releases it at low pO2; binding represents a sigmoidal curve. Hg responds to the Bohr effect, in which low pH, high CO2, high 2,3-BPG, and high temperature cause formation of salt bridges, leading to oxygen dumping.
- Mg: 1 O2 binds to Mg at high pO2 , but doesn’t get released until pO2 <5 mm Hg. Mg does not respond to the Bohr effect and its curve follows Michaelis-Menton.
4. Describe the concept of cooperative binding of O2 to hemoglobin and explain how it is quantified (the Hill Coefficient).
- As an additional O2 binds to an O2 binding site, it enhances the binding of more O2 to the other binding sites. There is a greater affinity for O2 as more O2 binds. The Hill Coefficient determines the extent (or absence) of cooperativity in molecules that bind O2. It is applicable for multisubunit molecules. When slope is >1, positive for cooperativity. When slope = 1, noncooperative molecule.
Hill coefficient for Hg = log (y/1-y) = nHlogpO2 – constant where y = saturation level 5. Compare and contrast the Hill Coefficient Plot for hemoglobin and myoglobin and explain the consequence of their difference.
- Hg has a slope = 2.2, which indicates that it is a cooperative molecule. Mg has a slope = 1, indicating that it is a noncooperative molecule due to the absence of cooperativity in this O2 binding molecule.
7. Define the Bohr Effect and demonstrate its impact on the Hb-oxygen dissociation curve.
- The Bohr Effect shows that low pH, high CO2, high 2,3-BPG, and high temperature result in decreased oxygen affinity, and thus oxygen dumping. The Bohr effect is dependent on cooperativity. Heterotrophic regulation of Hg by H+ & CO2 further increase O2–transporting efficiency. For instance, CO2 stabilizes deoxyHg to negatively charged carbanate groups, which are capable of salt bridge interactions.
8. Describe 2,3-bisphosphoglycerate and demonstrate its impact on the hb-oxygen dissociation curve.
- 2,3-BPG is highly anionic and interacts with positively charged side chains. Hg binds 1 molecule of BPG in central cavity. It increases salt bridge formation, stabilizes T state, and leads to decreased oxygen affinity. It is synthesized in RBCs from intermediate 1,3-BPG and is present in same [] as Hg. Its synthesis is promoted by low pO2 in peripheral tissues.
9. Describe the role of hemoglobin in CO2 transport and explain how the formation of carbaminohemoglobin facilitates oxygen transport.
- Hg has a role in CO2 transport. It stabilizes deoxyHg to form carbanate groups, which are negatively charged and capable of participating in salt bridge interactions.
[edit] Define: cooperativity and as it relates to hemoglobin
[edit] Graphs
[edit] Review the Bohr effect
The Bohr effect refers to the relationship between Hb's affinity for oxygen and the levels of CO2 and H+ in the blood. When CO2 levels are high (e.g., during exercise), the H+ concentration increases and the pH decreases because carbonic anhydrase converts CO2 to H+ and bicarbonate (HCO3-). This decreases Hb's affinity for oxygen , causing increased oxygen dumping (delivery of oxygen from the lungs to the tissues). Hb's affinity for oxygen is decreased because Hb binds H+ ions and CO2 molecules (in different sites than those to which oxygen binds), causing a conformational change that stabilizes the T conformation of Hb and facilitates the release of O2. Conversely, when Hb is oxygenated, its affinity for CO2 is reduced; this is called the Haldane effect.
source: http://content.nejm.org/cgi/content/full/338/4/239 (NEJM review) and Wikipedia
ASR
[edit] Effects with different pH
[edit] Structure of hemoglobin & myoglobin
[edit] Hemoglobin
[edit] Myoglobin
[edit] Different types of hemoglobin
There are at least five different types of hemoglobins. They differ in their subunit combinations. Each has a total of four subunits. There are two adult Hbs: HbA1 and HbA2. There is fetal hemoglobin, HbF. There are at least two embryonic Hbs: Hb Gower-1 and Hb Portland. The adult and fetus Hbs all have two alpha subunits.
source: Devlin
HbF binds O2 with more affinity (curve is shifted left) than adult Hb. This is necessary because the fetus blood must be able to take O2 from the maternal blood.
source: Wikipedia
JT
[edit] Different types of myoglobin
[edit] How hemoglobin and myoglobin transport O2 and CO2
[edit] How can hemoglobin be measured and used? (H1Ac)
Hemoglobin is measured by obaining a sample of blood, separating the red blood cells, lysing them, exposing the hemoglobin to a cyanide solution, and then conducting spectroscopic analysis on the solution to determine the density at a particular wavelength (540 nm usually) and thus the amount of hemoglobin present.
http://www.medicinenet.com/hemoglobin/article.htm
Here's an explanation of a total blood count which is just good to know:
http://www.medicinenet.com/complete_blood_count/article.htm
JT
[edit] Differences between hemoglobin and myoglobin
[edit] What other molecules can they bind?
Hb binds O2, CO2, glucose, and NO normally.
source: Devlin
CO competes with O2 to bind the heme protion of the Hb subunits and has an affinity 200 times greater. Other compounds that compete with O2 for heme binding are: CN-, SO, NO2, S^2-, and H2S. All of these compounds are toxic for this reason.
source: Wikipedia
JT
[edit] What dictates what they bind?
[edit] Can they bind O2 and CO2 at the same time?
[edit] Normal O2 and CO2 saturation levels
[edit] How it’s measured
A pulse oximeter relies on the fact that oxygenated and deoxygenated blood differ in their ability to absorb certain wavelengths of light. This machine must be placed on a semi translucent part of the body, such as a fingertip or earlobe, and the patient must have a pulse (I should hope they do).
source: Wikipedia
JT
[edit] Where it’s measured
Arterial saturation (Sa02) less than 90% is considered hypoxemia (insufficient oxygenation of the blood).
Venous saturation (SaO2) less than 60% indicates that the body is lacking oxygen.
source: Wikipedia of course
JT
[edit] How the levels differ and what it indicates
[edit] Hyperventilation
[edit] Hypoventilation
[edit] Effect of 2,3 bisphophoglycerate
[edit] Does it increase affinity or increase amount of hemoglobin?
It decreases the affinity of Hb for O2 because when 2,3-BPG binds Hb it stabilizes the T conformation. This causes Hb to release O2 to tissues. The concentration of 2,3-BPG increases in response to high altitude, chronic obstructive pulmonary disease, and chronic anemia.
source: Lippincott's
ASR
[edit] Graphs to explain regulation
[edit] Understand how difference situation cause graph shifts/movements
[edit] Pathologies associated with hemoglobin abnormalities
Sickle cell disease Hemoglobin C disease - single aa substitution in the Beta chain causes HbC; homozygous patients have a mild, chronic hemolytic anemia requiring no specific therapy Hemoglobin SC disease - two abnormal Beta chains but abnormal in a different way from each other; patient usually well until an infarctive crisis occurs, often following childbirth or surgery Methemoglobinemias - oxidation of the heme to F3+ state; can't bind O2; symptoms related to tissue hypoxia: anxiety, headache, and hyspnea Thalassemias - hereditary hemolytic diseases in which an imbalance occurs in the synthesis of globin chains (either alpha or beta)
source: Lippincott's
ASR
[edit] Discussion Notes
Case 3 – Globin family
Define: cooperativity and as it relates to hemoglobin
- Affinity for O2 increases as more O2 binds to Hb
- Each O2 bound increases affinity to a greater extent
- Hill Coefficient: log [(y)/(1-y)] = n(h) log pO2 - Const
- n(h) = slope
- If n(h) = 1, no cooperativity (ie myoglobin)
- If n(h)>1, positive cooperativity (ie hemoglobin)
- If n(h)<1, negative cooperativity
Review the Bohr effect
- Regulation of Hb affinity for O2 by [H+]
- Acidity lowers affinity [Dec pH, inc H+, inc CO2]
- Increased Temp lowers affinity
- H+ binds to Hb, causes formation of salt bridges that stabilize deoxy conformation (T-state)
Structure of hemoglobin & myoglobin
- Dimers (alpha, beta) held together by ionic bonds and H-bonds.
- T Form: Deoxy structure of Hb
- R Form: Oxy structure of Hb
- Disruption of ionic and hydrogen bonds leads to T Form to R Form change.
- Hb displays both concerted and sequential models of cooperativity
Different types of hemoglobin
- Adults:
- HbA (alpha 2, beta 2) 90% of total Hb
- HbA2 (alpha 2, delta 2), 2-5% of total Hb
- Fetal:
- HbF (alpha 2, gamma 2), synthesized predominatly during fetal development
- <2% of total Hb in adults
Myoglobin
- Higher affinity for O2 than Hb
- Displays Michaelis-Mentin Kinetics
- Mb stores O2 in skeletal muscle
- Unloads O2 at very low [O2]
Normal O2 and CO2 saturation levels How it’s measured
- Pulseoximetry (measures light absorption; difference between systole, diastole)
Where it’s measured
- Skin surface
How the levels differ and what it indicates
- Hypoxemia: arterial saturation <90% (measured with pulseoximeter); venous saturation <70% (measured with special catheter); AV-O2 difference is a measure of oxygen consumption (normal difference is 25%)
How hemoglobin and myoglobin transport O2 and CO2
- O2 binds to heme group
- CO2 binds at another site
- CO is a potent competitive inhibitor of O2 (binds to heme group)
- Haldane Effect: how partial pressure of oxygen affect Hb affinity to CO2.
- When O2 is low, Hb has increased affinity for CO2
- Binding of CO2 facilitates unloading of O2
- Carbaminohemoglobin: CO2 binding to Hb. Make vein appears blue
How can hemoglobin be measured and used? (H1Ac)
- Measures blood glucose concentration over lifespan of RBC (120 days)
- Hemoglobin:Hematocrit ratio = 1:3; 12g Hb = 36% Hematocrit
Differences between hemoglobin and myoglobin
- Hemoglobin = Transport protein; Tetramer; Demonstrates cooperativity
- Myoglobin = Storage protein; Monomer; Does not demonstrate cooperativity
What other molecules can they bind?
- Hb binds oxygen, nitric oxide (NO), hydrogen, nitrous oxid (N2O) [converts hb to methemoglobin], nitrogen dioxide (NO2) [competitive inhibitor], carbon dioxide, 2-3 BPG
- Mb binds O2
- If trauma and urine is red, look under microscope; Presence of RBC indicates hemoglobinurea, if no RBC indicates myoglobinurea.
Effect of 2,3 bisphophoglycerate
- Decreases affinity of Hb for O2
- Forms salt bridges with lysine and histidiene residues
- Binds to a positively charged cavity formed by the beta-chains of deoxyhemoglobin
- 2,3 BPG is made from an intermediate of glycolysis
- 2,3 BPG production is increased under conditions of low oxygen partial pressure. This facilititates oxygen release from hemoglobin into the tissues.