Chapter 5 Flashcards
Oxygen transport proteins
- myoglobin and hemoglobin are part of a large family of oxygen transport proteins
Ligand
a molecule that reversibly binds a protein
Globin family of proteins
- present in all branches of life (archae, bacteria, eukaryotes, prokaryotes)
- defined by the highly conserved globin fold - example of a structural motif
Why do we need proteins to transport O2?
- O2 cannot easily diffuse in mammals
- O2 is a diradical (reactive), leads to reactive oxygen series and damage biomolecules
What is the function of myoglobin?
- binds O2
- facilitates diffusion of O2 into muscle
What is the function of Hemoglobin?
- binds O2
- transports O2 through the body in blood
- picks up O2 from lungs and disperse to tissues
Structure of Myoglobin
- monomeric (1 chain, 1 subunit)
- 1 heme
Structure of Hemoglobin
- tetrameric (heterotrimer)
- 4 hemes
Prosthetic Group
non amino acid group that is bound to the protein
- many proteins contain prosthetic groups that support their function
What is the prosthetic group in myoglobin/ hemoglobin?
Heme group
What does a heme group do?
- A heme group is permanently bound to each globin domain and is responsible for O2 binding and the red color of Mb/Hb
When is a heme group added?
the heme group is added to Mb/Hb post-translationally and is responsible for binding O2
What does a heme group contain?
- a protoporphyrin ring
- Fe 2+ atom
What are the six bonds that “coordinate” iron?
-four nitrogens in the porphyrin ring, - one nitrogen in the proximal Histidine, and the O2
What is the ligand for myoglobin/hemoglobin?
oxygen
What binds iron?
The proximal histidine
What hydrogen bonds to the O2?
the distal histidine
Carbon monoxide poisoning
- CO bind to hemoglobin 40-times more tightly than O2
- O2 has a non linear bond while CO has a linear bond which is strongest allowing CO to outcompete O2
- Poisoning is a result of CO outcompeting O2
Equilibrium expression that describes the reversible binding of a protein (P) to a ligand (L)
P+L ⇌ PL
Ka=[PL]/[P][L]
Kd: Dissociation Constant
= 1/ Ka = [P][L]/[PL]
- smaller Kd= tighter binding
Fractional Saturation Y
- Y= [PL]/[P]+[PL]
= (bound active sites)/(total active sites) - Y= [L]/[L]+[Kd] ; form of a rectangular hyperbola
[L]=pO2 (partial pressure of O2)
What determines binding affinity?
chemical bonds and the intermolecular forces between ligand and its binding site
What are the pros/cons of high affinity binding?
- If binding is to high then it can’t unbind to complete its function
- we want reversible binding
- we need it to bind tight enough to do its job
Subunit Composition of Hemoglobin
-Hemoglobin tetramer: α2β2
-multiple globin genes
- fetal hemoglobin: α2γ2 (binds more tightly to oxygen)
fetal hemoglobin
- α2γ2
- binds more tightly to oxygen so that babies can get oxygen from their mother
What is the benefit of more subunits?
- allows for more regulation
- “communication” between the subunits allows for more cooperative binding of O2
Cooperativity
binding of one ligand increasing the affinity for binding subsequent ligands
- O2 binding at one subunit of Hb must change the other binding sites ( increasing affinity)
What are the two distinct conformations or state of hemoglobin?
- R-State: oxyhemoglobin (~active) O2 bound
- T-State:deoxyhemoglobin (~inactive) unbound
What influences the balance between the R and T state of hemoglobin?
O2 and effectors
What stabilizes the T state?
stabilized by subunit interface interactions
What happens to Fe 2+ when O2 binds?
the iron atoms shrinks
What happens of the protoporphyrin ring when O2 binds?
concave shape —> flattens out
What happens to the helix attached to histidine F8 (proximal) when O2 binds?
shifts the whole alpha helix/ causes conformational change throughout
What is low O2 associated with?
low-affinity/T-State
What is high O2 associated with?
high-affinity/R-State
Homotropic modulator
- O2 binding favors R state
- O2 is the ligand
Heterotrophic modulators
- Heterotropic= not the ligand
- H+ and CO2 regulate and are all transported by Hb
- both favor the T state
Bohr Effect
the effect of pH and CO2 on the binding and release of O2 by hemoglobin
How is H+ carried?
“carried via protonation of His146 (β) (as well as other His residues)
How is CO2 removed?
its covalently attached to the N-terminus of each chain
- both alpha and beta subunits
- NH3+ converted to carbamate
- carried to lungs and removed
2,3-Biphosphoglycerate (BPG)
- produced in respiring tissues
- binds the central cavity of hemoglobin, stabilizing the T state
What are antibody functions inside the body?
- involved in the immune response (humoral and cellular)
- recognize and bind foreign agents and recruit immune cells
How are antibodies made by the body?
- produced in B lymphocytes or B cells
- one B cell produces one kind of antibody
- antibodies make up 20% of your total blood protein
- at anytime there are 100,000,000 different antibodies circulating
Variable regions
antigen binding sites are created by random shuffling of the immunoglobulin genes
- is what gives rise to different types of antibodies
5 Classes of Immunoglobulins in Vertebrates
- characterized by heavy chain
1. alpha for IgA: mucus, tears, saliva
2. delta for IgD: on B cell receptors
3. epsilon for IgE: allergies
4. Gamma for IgG: late defense
5. mu for IgM: early defense
Immunoglobulin G Structure
- two heavy chains and two light chains
- antigen binding site varies
- bottom part binds to receptors on b-cells
- Top part: (FAB) fragment that does antigen binding
- Bottom part: (FC) constant fragment
Antibody/Immunoglobulin/Ig
A protein generated against, and capable of binding specifically to, an antigen
Antigen
A molecule capable of eliciting the synthesis of a specific antibody in vertebrates
Epitope
An antigenic determinant; the particular chemical group or groups in a macromolecule (antigen) to which a given antibody binds
Induced fit
ligand binds and binding site form fits around ligand (hand and glove)
What influences antigen binding/ affinity?
IMFs between the epitope and antigen binding site on IgG
Polyclonal Antibody
population of B cells that produce a bunch of IgG against 1 antigen
- strong but less specific binding
Monoclonal Antibody
1 B cell that makes 1 antibody
- weaker binding but very specific
Examples of antibodies in research
- Western blots
- ELISAs
- Immunoprecipitation
