Ligands bind at specific sites on a protein called...
binding sites
Proteins must be correctly folded to have....
proper function
Proteins must also interact with other molecules and these interactions are dictated by...
protein structure
Reversible bind of ligands is
any kind of molecule that reversibly binds to a protein
Protein structure forms...
the binding site
Which characteristics of the binding site dictate ligand binding?
Complementary Characteristics like
Size, Shape, Charge, and Hydrophobicity/ Hydrophilicity
Give an Example of Protein-Ligand Interactions
Oligopeptide-binding protein, OppA
Binding of the KWK tripeptide in the Xanthomonas citri OppA binding site
KWK modeling disclose polar interactions
4 Reasons why Myoglobin and Hemoglobin are studied
1. Most studied, best understood proteins
2. first proteins whose 3D structures were determined
3. Both are oxygen binding proteins
4. Illustrate a central biochemical process-- reversible binding of a ligand to a protein
Hemoglobin participates in oxygen transport by....
taking it from the lungs to the tissues. Binds O2 in the lungs and releases it in the tissues.
Myoglobin participates in oxygen transport by
storing O2 for deliver in rapidly respiring muscle tissues. Binds O2 when not needed by tissues, releases it when necessary
3 similar characteristics of Myoglobin and Hemoglobin
1. they are two important oxygen proteins (not enzymes)
2. two of the most studied proteins in nature
3. offers evidence of how protein structure relates to function
Remember: Hemoglobin model for protein quaternary structure and allosteric functions
4 Myoglobin structural characteristics include...
1. contains 153 amino acids
2. Molecular Weight of 17.2 kD
3. Globular structure has 8 alpha helical segments with connecting segments
4. Contains a heme prosthetic group in a protoporphyrin ring with an Fe 2+ bound
What is a protoporphyrin ring?
Contains an Fe2+ which is important for binding oxygen, and is classified as a heme prosethetic group in myoglobin and hemoglobin
Hemoglobin structural characteristics
1. globular structure has 2 alpha, 2 beta polypeptide chains
2. Molecular Weight of 64.45 kD
3. Contains a Heme prosethetic group which is a protoporphyrin ring contaiing Fe 2+ important for O2 binding
Myoglobin functions as...
an oxygen storage and transport proteins in muscles
Hemoglobin functions in...
oxygen delivery via blood vessels
Which common structural motif is shared by myoglobin and hemoglobin?
Globin Fold (8 alpha helices + heme)
The polypeptide chains of myoglobin and hemoglobin fold around a...
prosthetic group-heme
Where is the heme located in myoglobin (monomer) and hemoglobin (tetramer)?
in a hydrophobic pocket between helices
What is myoglobin's role in O2 binding?
facilitates oxygen diffusion to muscle and rapidly respiring tissue
O2 diffusion is limited from capillaries to tissues but myoglobin acts as a molecular bucket to increase O2 diffusion
(Very important in aquatic mammals for higher oxygen storage needs un
4 structural properties of Myoglobin
1. Heme group: protoporphyrin ring with Fe 2+ bound
2. Spherical (globular)
3. 8 helices, A-H
4. bends between helices: AB, CD, EF, GH
Why does O2 need a heme group?
O2 is not very soluble in aqueous solutions, and its solubility and transportability is increased when bound to a soluble carrier such as transition metals. (Amino acids do not reversibly bind to O2)
Describe the composure of a Heme group: protoporphyrin ring.
There are 4 porphyrin nitrogen bound to a Fe2+ which is also bound to a N from His F8 residue (proximal His) These are 5 ligands. The 6th ligand is O2. A His E7 (distal His) hydrogen bonds to O2.
Iron is used to transport O2
Val E11 and Phe CD1 hold the h
What happens when O2 binds to iron?
It brings the iron up into the plane of the protoporphyrin ring.
Describe oxygenation of Heme and the different colors of blood
Binding of O2 to Fe 2+ alters heme complex electronic state which changes the color
From dark purple (venous blood) to scarlet blood( arterial blood- more oxygenated)
What happens if Fe II is oxidized to Fe III?
prevents binding of O2
heme complex of Fe III is brown, called methemyoglobin-dried blood
What can also bind to Fe(II) heme complex?
CO, NO, H2S, all of which have higher binding affinities, or SMALLER Kds so they are toxic
How is carbon monoxide poisoning a threat?
CO has 20,000 smaller Kd than O2, meaning is has a significantly higher binding affinity for Fe(II) than O2 in myoglobin
Why can you survive for a small period of time when carbon monoxide is present?
CO binds less readily with heme which already has an O2 bound because of the His E7 (distal His) bound to O2 which creates steric interference. CO binds tighter to unbound myoglobin
Describe binding
Is reversible P+L >< PL
Can be described with Kd = [P][L]/[PL]
which is inverse of Ka (association constant)
Large Kd, protein feels low affinity for its ligand
Small Kd, the protein adores its ligand and is less likely to release it.
Formula for fraction of ligand binding sites that are occupied
Fraction of Ligand Binding Sites Occupied = [PL]/ [PL] + [P]
Myoglobin is a protein (not an enzyme) and it's ligand is...
O2
Henry's Law
Quantity of gas in liquid is proportional to partial pressure of gas above liquid
[O2] proportional to pO2
Describe the dissociation of the protein-ligand complex for myoglobin
MbO2 >< Mb +O2
Kd = [Mb][O2]/ [MbO2]
or
Kd= [Mb]pO2/ [MbO2]
What is Yo2 and what are the ranges?
Yo2 is fraction of O2 binding sites occupied
ranges from 0 (no bound O2) to 1 (all binding sites are filled)
Fractional Saturation for General Protein-Ligand Complexes and Myoglobin
Fractional Saturation= [L]/ Kd + [L]
Myoglobin Yo2= pO2/ Kd + pO2
or Yo2= [O2]/ Kd + [O2]
Describe percent oxygen saturation or fractional percent of O2 binding
Fractional Percent of Oxygen Saturation= Number of binding sites occupied/ total binding sites
[MbO2]/ [Mb] + [MbO2]
describe p50
where Kd is equal to concentration of ligand in which half of sites are occupied
For myoglobin, this identifies what the oxygen pressure is when myoglobin is 50% saturated with O2
Describe p50, Kd, and affinity relationship
High p50, high Kd, means low protein affinity for ligand
Low p50, low Kd, means protein adores ligand and clings to it
Kd=p50 so Y= pO2/ (p50 + pO2)
Describe O2 saturation curve for Myoglobin
Has a low p50, so half saturation is obtained quickly
Hyperbola curve
2 points on curve are obvious: half Saturation and full saturation (never attained)
The p50 value for Myoglobin is...
2.8 torr
Arterial blood has a pO2 ([O2]) or 100 torr. What is the fractional saturation of Myoglobin?
Remember, myoglobin p50 (half saturation) is 2.8 torr.
Fractional Percent Saturation= pO2/ Kd + pO2
=100 torr/ (2.8 torr + 100 torr)= 0.97 which means 97% of Myoglobin has O2 bound to it
The binding is considered weaker when it takes a...
greater oxygen pressure (larger p50) to reach half saturation
On a O2 saturation curve, x axis is pO2 and y axis is fractional saturation
Describe Hemoglobin structure
1. alpha2,beta2 tetramer (4 myoglobin like polypepties) formed from a diner of alphabeta protomers
2. alpha and beta subunits are structurally like myoglobin
3. has 4 oxygen binding sites
4. Oxygen binding alters the structure of entire hemoglobin
5. sigm
Describe oxygen binding in hemoglobin
must be able to bind oxygen in lungs (high pO2, forces binding)
Hb + O2 yields HbO2 (OxyHb)
releases oxygen in capillaries (low pO2, encourages release)
HbO2 yields Hb + O2
What would happen if Hemoglobin has a high affinity for O2 (low Kd/p50) like Myoglobin?
would not want to release O2 to capillaries. Hemoglobin has a much higher p50 (lower afinity) compared to Myoglobin.
Hemoglobinr has a sigmoidal (indicates copperativity) binding curve while with 4 binding sites myoglobin's is a rectangular hyperbola and
Hemoglobin affinity for O2 becomes greater when
oxygen is progressively bound, creating stronger affinity
must be able to bind in lungs and release in capillaries
Myoglobin must constantly have a low p50 (high affinity) to store O2 until needed
If myoglobin had a p50 that was the same as hemoglobin's p50,
myoglobin would no longer be a good O2 storage protein because it's affinity for O2 was decreased
Why is hemoglobin saturation curve sigmoidal?
4 binding sites for O2, when oxygen binds it alters the structure in a quaternary shift. Affinity increases as each binding site is occupied
In any binding system, a sigmoidal shape indicates...
cooperativity
what is cooperativity
the binding of a ligand at one site affects binding of Ligands at another site
If Kd>>1, it favors
[P][L]
in P+L >< PL
If Kd<<1, it favors
[PL]
in P+L >< PL
How does hemoglobin bind an n, number of O2 molecules in a cooperative fashion?
using positive cooperativity
Hb +nO2 yields Hb(O2)n
Hb binding O2 at one site increases affinity for O2 at another-- cooperative process
Define Y, fractional saturation of Hemoglobin
Y= pO2^4/ pO2^4 +Kd
or Y= [O2]^4/ [O2]^4 +Kd
At n=1, hemoglobin does not show
positive cooperativity
Y= pO2^n/ pO2^n +Kd
n=1-4 lines are averaged into a sugmoidal curve
Define Hill Equation
used for cooperative ligand binding
Hb(O2)n >< Hb +nO2
Y= pO2^n / pO2^n + p50^n
n is the Hill constant/coefficient
Describe differences in the Hill Constant
n=1, non cooperative binding, hyperbolic
n>1, positive cooperativity- sigmoidal
n<1, negative cooperativity- sigmoidal
Hill Equation and Plot FULL
log (y'/1-y') = nlogpO2 - nlogp50
When y (log y'/1-y') is plotted against x (log pO2)
Region of max slope is n
logp50= x intercept
When y (log y'/1-y') is 0
then the y' is 0.5
nlogpO2 = nlogp50 or pO2 = p50
max slope is n
Myoglobin hill plot slope is...
1 because only 1 binding site and is non cooperative
p50= x intercept
oxygen transport torrs
158 inspired air
100 aveolar air
90 arterial blood
capillary 40
interstitial 30
cytosol 10
Cooperativity is designed to...
release O2 at low pressures
weakest afffinity at n=1 and strongest affinity at n=4
Structure of hemoglobin more detailed
1. alpha2beta2 tetramer
2. quaternary structure is dimer of alphabeta dimers
3. alphabeta dimers associate bia hydrophobic/ ionic interactions and hydrogen bonds (not affected by pH)
4.The dimer of alphabeta dimers (tetramer) only has ionic interactions a
Describe conformational changes caused by oxygen binding
Without O2 bound, Fe II is out of heme plane, with O2, is in heme plane, pulling His F8 along with it.
The F helix moves which acts as a trigger that allows for a conformational change creating greater affinity for O2 at other binding sites in hemoglobin.
Cooperativity of binding multiple O2 molecules in hemoglobin results from a...
structural change!
Describe two stable states of hemoglobin
R state: the state oxygen loves and clings to the most
T state: predominates in the absence of oxygen, but oxygen can bind to it...
T state is deoxyhemoglobin
R state is oxyhemoglobin
When deoxy-hemoglobin crystals are exposed to oxygen... they
shatter! proof of structural change
What happens when oxygen binds to hemoglobin in terms of degrees?
one alphabeta pair moves relative to the other by 15 degrees--induced by a movement of Fe II by 0.029 nm
T-state (deoxy Hb) is stabilized by a series of______ that lie at the intersection between the two alphabeta protomers
?
The structural shift from T state to R state predominantly affects what?
the quaternary structure of the alphabeta protomers
Detail the T to R conformation shift
1. His F8 moves with Fe2+ toward heme
2. His F8 packed away in hydrophobic pocket reorients Helix F when moved
3. Helix F moves, producing quaternary shift in the alpha1C-Beta2FG contacts
4. alpha1-beta1 and alpha2-beta2 interactions remain unchanged, so
pO2 for venous blood
30 torr
pO2 for arterial blood
100 torr
p50 for Myoglobin
2.8 torr- no cooperativity
p50 for Hemoglobin
Average is 26 torr- shows cooperativity
1st binding p50 is 30 torr
2/3rd binding p50 is ~12 torr
4th binding p50 is 0.3 torr
T to R transition in hemoglobin must occur simultaneously at...
the interface between the two alphabeta protomers
Describe 4 binding site steps in hemoglobin
1. First binding, low affinity T, no cooperativity
2. Second binding induces T to R, interface destabilized enough for T to R to transition
3. 3rd Binding easier- High Affinity for R state
4. Last binding occurs readily- Very high Affinity for R state- no
Conclusions about Cooperativiy and the Quaternary shift (3)
1. Binding of one O2 results in strain on T-state that tears away C-term ion pairs
2. quaternary shifts to R state increasing binding site's love (affinity) for O2
3. O2 binding become more efficient
Oxygenation causes the F helix of one alpha beta dimer to...
shift relative to the C-term of the opposite alphabeta dimer
Interactions in the structure of hemoglobin
1. dimer of dimers (alphabeta) makes up a tetramer
2. the alpha beat dimer is stable due to hydrophobic interactions at dimer interface working with ionic interactions and hydrogen bonds
3. the alpha1beta1 dimer alpha2beta2 dimer interface forming the tet
4 concepts of quaternary shift and O2 binding
1. T state (deoxyHb) stabilized by ionic interactions
2. Quaternary shift is the movement of residues in the alpha1beta1 dimer- alpha2beta2 dimer interface
3. R state (oxyHb)- possible due to energy harnessed from Fe2+ bond with O2 during 1st O2 binding
4
Describe changes in ionic interactions during T-R transition for T-state
The formation of ion pairs increases the pK values of the group.
Increase pK, decrease acidity (increase pH). Therefore will cling to, and hold hydrogens. MORE BASIC
Describe changes in ionic interactions during T-R transition for R-state
interaction of ion pairs are absent causing a decrease in pK (increasing acidity, lowering pH). Will readily release, throw away protons. Donate protons
Hb releases ____ ____ for each O2 it binds
0.6 H+
Effectors on Hemoglobin's affinity for O2
[O2]
[H+] - Bohr Effect
[CO2]
[2,3 Bisphophoglycerate] - BPG
How does an increase in the [H+] (more acidic, lower pH) affect the binding curve of Hemoglobin for O2?
it moves the Hb binding curve to the right, with a higher p50 value (lower affinity for O2)
For each O2 that binds to hemoglobin, what happens to the environment?
hemoglobin releases ~0.6 H+ and pH decreases (increases acidity)
Decrease in pH (increase in H+) does what to Hemoglobin's affinity?
Decreases it
Increase in pH (decrease in H+) does what to Hemoglobin's affinity?
Increases affinity (more basic)
Christian Bohr
observed the decrease in O2 binding affinity of hemoglobin in response to a decrease in pH
Bohr Effect
Bohr Effect
has important physiological functions in transporting O2 from lungs to respiring tissue
related to blood buffering system- important because a constant biological pH must be maintained for physiological reactions
Blood buffering system
Carbonic Acid (H2CO3) / bicarnonate (HCO3-)
not the only buffer
CO2 (g) >< CO2 (aq)
CO2 (aq) + H2O (l) >< H2CO3 (aq)
H2CO3 (aq) >< H+ (aq) + HCO3- (aq)
----------------------------------------------------
Total Equation: CO2 (g) + H2O (l) >< H+ (aq) + HCO
What happens to the blood buffering system if pH decreases?
If pH decreases, more acidic, so there is an increase in [H+] from metabolic reactions
Equilibrium shifts towards H2CO3
H+(aq) + HCO3- (aq) yields H2CO3 (aq)
and
H2CO3 loses water to produce CO2, expelled by lungs
H2CO3 (aq) yields CO2 (aq) + H2O (aq)
CO2
What happens to the blood buffering system if pH increases?
becomes more basic so there is a decrease in [H+]. Breathing adjusts to introduce more CO2 into blood from lungs to allow ultimate conversion of H2CO3
CO2 (g) yields CO2 (aq)
CO2 (aq) + H2O (aq) yields H2CO3 (aq)
What enzyme accelerates the diffusion of CO2 from respiring tissue back to the lungs?
carbonic anhydrase in red blood cels
What does hemoglobin do with H+ in the T-state
Binds to it.
What does hemoglobin do with H+ in the R state?
Releases it
How does increase pO2 affect uptake/ release of H+ for hemoglobin?
Increase pO2, H+ from Hb is taken by HCO3- to produce H2CO3 yields H2O + CO2
How does decreasing pO2 affect uptake/release of H+ for hemoglobin?
Decrease pO2, H+ from H2CO3 is taken by Hb to produce HCO3-
Show the raw equation for CO2 being expelled in lungs, and HCO3- in tissues preparing to travel back to the lungs
CO2 +H2O >< H+ + HCO3-
What is carbamation
Hemoglobin acts as a CO2 transporter
How is CO2 moved about the body?
1. as HCO3- in blood
2. binds to N-terminal amino acid groups of hemoglobin subunits to form carbamates
carbamates
CO2 bound to N-terminal amino acid groups of hemoglobin subunits
Which of the 2 states of Hb binds CO2 better?
T state
remember, H+ is released upon binding.
Why does the R form shift to T form and release O2?
because T form binds CO2 better, and H+ is released upon binding
CO2 binds to N terminus of Hb liberating a H+ creating the Bohr effect with decreases Hb's affinity for O2 (decrease in pH, decrease in affinity)
Affects of Carbamation
1. aids in transport of CO2 TO the lungs
2. increases Bohr effect by releasing H+ when the CO2 binds to the N terminus of amino acids in Hb
3. Carbamates can form ion pairs stabilizing T state (deoxy Hb)
How much does Hemoglobin aid the transportation of CO2?
~50% transported by the blood for exhaling out of the lungs by hemoglobin
Which state does 2,3-Bisphosphoglycerate (BPG) bind in hemoglobin? How does it effect the binding affinity for O2?
T-state (deoxy Hb)
Decreases affinity for O2
Decribe oxygen saturation of hemoglobin in artieries and veins
Arteries (have a pO2 ~100 torr) allow Hb to become 95% saturated with O2
Veins (have a pO2 ~30 torr) allow Hb to be 55% saturated with O2
How does the presence of 2,3- Biphosphoglycerate (BPG) help Hemoglobin?
Presence of BPG helps Hb unload its O2 into tissues
How does BPG help Hb release O2 from its binding sites?
BPG in T-state (deoxyHb) binds at central cavity which narrows during the T to R quaternary shift releasing the BPG
What does fetal hemoglobin have small amounts if none of?
BPG, it does not readily allow 2,3-biphosphoglycerate to bind
Important for fetus to obtain O2 from mom's circulatory system
Joseph Barcroft
Observed highly purified, stripped Hemoglobin binds O2 with a higher affinity than Hb in whole blood due to allosteric effects. CO2 and BPG are both negative alloseteric effects that diminish hemoglobin's affinity for O2.
Allostery
the binding of a ligand at one site affects the binding of ANOTHER LIGAND at another site
Explain CO2 and BPG affects on binding affinity for O2 in hemoglobin.
CO2 and BPG bind at sites other than O2 binding sites and decrease affinity for O2
Explain Symmetry model of allostery
Hb is capable of existing in T or R state.
Ligands can bind subunit in either conformation and only the conformational change alters affinity for a ligand. The conformational change when all subunits are the same conformation.
Problems:
1. conformational
Explain Sequential model of Allostery
Ligand binding induces conformational change in the subunit it binds and cooperative interactions result
Cooperative Interactions change neighboring subunits that influence its affinity for ligand
Allostery models of O2 binding in Hemoglobin realistically involves
both symmetry and sequential.
Change in T to R states is concerted-symmetry model
Ligand binding in T-state results in small tertiary structural changes that cooperatively induce Hb's final quaternary shift- sequential model
Hemolytic anemia
Mutation in Hb
deficiency in red blood cells, compromises O2 delivery to tissue
Cyanosis
Mutation in Hb. Methemoglobin predominates from mutation (Fe II to Fe III) reducing O2 binding cooperativity, bluish skin results
Polycythemia
Mutation in Hb
increases O2 affinity for Hb- increased red blood cells to compensate for less O2 being released
ruddy complexion
Sickle-cell anemia
Mutations in Hb
single amino acid substitutions
carried by 25% african americans, Hb S
heterozygous suffer mildly
homozygous get sick- Mis-shaped red blood cells can't pass through capillaries
Why are african americans prone to sickle cell amenia genes?
Hb S protects against malaria- heterzygous gets protection
Adaptive advantage like Cystic Fibrosis in europeans making them more resistent to cholera
Ligands bind at specific sites on a protein called...
binding sites
Proteins must be correctly folded to have....
proper function
Proteins must also interact with other molecules and these interactions are dictated by...
protein structure
Reversible bind of ligands is
any kind of molecule that reversibly binds to a protein
Protein structure forms...
the binding site
Which characteristics of the binding site dictate ligand binding?
Complementary Characteristics like
Size, Shape, Charge, and Hydrophobicity/ Hydrophilicity
Give an Example of Protein-Ligand Interactions
Oligopeptide-binding protein, OppA
Binding of the KWK tripeptide in the Xanthomonas citri OppA binding site
KWK modeling disclose polar interactions
4 Reasons why Myoglobin and Hemoglobin are studied
1. Most studied, best understood proteins
2. first proteins whose 3D structures were determined
3. Both are oxygen binding proteins
4. Illustrate a central biochemical process-- reversible binding of a ligand to a protein
Hemoglobin participates in oxygen transport by....
taking it from the lungs to the tissues. Binds O2 in the lungs and releases it in the tissues.
Myoglobin participates in oxygen transport by
storing O2 for deliver in rapidly respiring muscle tissues. Binds O2 when not needed by tissues, releases it when necessary
3 similar characteristics of Myoglobin and Hemoglobin
1. they are two important oxygen proteins (not enzymes)
2. two of the most studied proteins in nature
3. offers evidence of how protein structure relates to function
Remember: Hemoglobin model for protein quaternary structure and allosteric functions
4 Myoglobin structural characteristics include...
1. contains 153 amino acids
2. Molecular Weight of 17.2 kD
3. Globular structure has 8 alpha helical segments with connecting segments
4. Contains a heme prosthetic group in a protoporphyrin ring with an Fe 2+ bound
What is a protoporphyrin ring?
Contains an Fe2+ which is important for binding oxygen, and is classified as a heme prosethetic group in myoglobin and hemoglobin
Hemoglobin structural characteristics
1. globular structure has 2 alpha, 2 beta polypeptide chains
2. Molecular Weight of 64.45 kD
3. Contains a Heme prosethetic group which is a protoporphyrin ring contaiing Fe 2+ important for O2 binding
Myoglobin functions as...
an oxygen storage and transport proteins in muscles
Hemoglobin functions in...
oxygen delivery via blood vessels
Which common structural motif is shared by myoglobin and hemoglobin?
Globin Fold (8 alpha helices + heme)
The polypeptide chains of myoglobin and hemoglobin fold around a...
prosthetic group-heme
Where is the heme located in myoglobin (monomer) and hemoglobin (tetramer)?
in a hydrophobic pocket between helices
What is myoglobin's role in O2 binding?
facilitates oxygen diffusion to muscle and rapidly respiring tissue
O2 diffusion is limited from capillaries to tissues but myoglobin acts as a molecular bucket to increase O2 diffusion
(Very important in aquatic mammals for higher oxygen storage needs un
4 structural properties of Myoglobin
1. Heme group: protoporphyrin ring with Fe 2+ bound
2. Spherical (globular)
3. 8 helices, A-H
4. bends between helices: AB, CD, EF, GH
Why does O2 need a heme group?
O2 is not very soluble in aqueous solutions, and its solubility and transportability is increased when bound to a soluble carrier such as transition metals. (Amino acids do not reversibly bind to O2)
Describe the composure of a Heme group: protoporphyrin ring.
There are 4 porphyrin nitrogen bound to a Fe2+ which is also bound to a N from His F8 residue (proximal His) These are 5 ligands. The 6th ligand is O2. A His E7 (distal His) hydrogen bonds to O2.
Iron is used to transport O2
Val E11 and Phe CD1 hold the h
What happens when O2 binds to iron?
It brings the iron up into the plane of the protoporphyrin ring.
Describe oxygenation of Heme and the different colors of blood
Binding of O2 to Fe 2+ alters heme complex electronic state which changes the color
From dark purple (venous blood) to scarlet blood( arterial blood- more oxygenated)
What happens if Fe II is oxidized to Fe III?
prevents binding of O2
heme complex of Fe III is brown, called methemyoglobin-dried blood
What can also bind to Fe(II) heme complex?
CO, NO, H2S, all of which have higher binding affinities, or SMALLER Kds so they are toxic
How is carbon monoxide poisoning a threat?
CO has 20,000 smaller Kd than O2, meaning is has a significantly higher binding affinity for Fe(II) than O2 in myoglobin
Why can you survive for a small period of time when carbon monoxide is present?
CO binds less readily with heme which already has an O2 bound because of the His E7 (distal His) bound to O2 which creates steric interference. CO binds tighter to unbound myoglobin
Describe binding
Is reversible P+L >< PL
Can be described with Kd = [P][L]/[PL]
which is inverse of Ka (association constant)
Large Kd, protein feels low affinity for its ligand
Small Kd, the protein adores its ligand and is less likely to release it.
Formula for fraction of ligand binding sites that are occupied
Fraction of Ligand Binding Sites Occupied = [PL]/ [PL] + [P]
Myoglobin is a protein (not an enzyme) and it's ligand is...
O2
Henry's Law
Quantity of gas in liquid is proportional to partial pressure of gas above liquid
[O2] proportional to pO2
Describe the dissociation of the protein-ligand complex for myoglobin
MbO2 >< Mb +O2
Kd = [Mb][O2]/ [MbO2]
or
Kd= [Mb]pO2/ [MbO2]
What is Yo2 and what are the ranges?
Yo2 is fraction of O2 binding sites occupied
ranges from 0 (no bound O2) to 1 (all binding sites are filled)
Fractional Saturation for General Protein-Ligand Complexes and Myoglobin
Fractional Saturation= [L]/ Kd + [L]
Myoglobin Yo2= pO2/ Kd + pO2
or Yo2= [O2]/ Kd + [O2]
Describe percent oxygen saturation or fractional percent of O2 binding
Fractional Percent of Oxygen Saturation= Number of binding sites occupied/ total binding sites
[MbO2]/ [Mb] + [MbO2]
describe p50
where Kd is equal to concentration of ligand in which half of sites are occupied
For myoglobin, this identifies what the oxygen pressure is when myoglobin is 50% saturated with O2
Describe p50, Kd, and affinity relationship
High p50, high Kd, means low protein affinity for ligand
Low p50, low Kd, means protein adores ligand and clings to it
Kd=p50 so Y= pO2/ (p50 + pO2)
Describe O2 saturation curve for Myoglobin
Has a low p50, so half saturation is obtained quickly
Hyperbola curve
2 points on curve are obvious: half Saturation and full saturation (never attained)
The p50 value for Myoglobin is...
2.8 torr
Arterial blood has a pO2 ([O2]) or 100 torr. What is the fractional saturation of Myoglobin?
Remember, myoglobin p50 (half saturation) is 2.8 torr.
Fractional Percent Saturation= pO2/ Kd + pO2
=100 torr/ (2.8 torr + 100 torr)= 0.97 which means 97% of Myoglobin has O2 bound to it
The binding is considered weaker when it takes a...
greater oxygen pressure (larger p50) to reach half saturation
On a O2 saturation curve, x axis is pO2 and y axis is fractional saturation
Describe Hemoglobin structure
1. alpha2,beta2 tetramer (4 myoglobin like polypepties) formed from a diner of alphabeta protomers
2. alpha and beta subunits are structurally like myoglobin
3. has 4 oxygen binding sites
4. Oxygen binding alters the structure of entire hemoglobin
5. sigm
Describe oxygen binding in hemoglobin
must be able to bind oxygen in lungs (high pO2, forces binding)
Hb + O2 yields HbO2 (OxyHb)
releases oxygen in capillaries (low pO2, encourages release)
HbO2 yields Hb + O2
What would happen if Hemoglobin has a high affinity for O2 (low Kd/p50) like Myoglobin?
would not want to release O2 to capillaries. Hemoglobin has a much higher p50 (lower afinity) compared to Myoglobin.
Hemoglobinr has a sigmoidal (indicates copperativity) binding curve while with 4 binding sites myoglobin's is a rectangular hyperbola and
Hemoglobin affinity for O2 becomes greater when
oxygen is progressively bound, creating stronger affinity
must be able to bind in lungs and release in capillaries
Myoglobin must constantly have a low p50 (high affinity) to store O2 until needed
If myoglobin had a p50 that was the same as hemoglobin's p50,
myoglobin would no longer be a good O2 storage protein because it's affinity for O2 was decreased
Why is hemoglobin saturation curve sigmoidal?
4 binding sites for O2, when oxygen binds it alters the structure in a quaternary shift. Affinity increases as each binding site is occupied
In any binding system, a sigmoidal shape indicates...
cooperativity
what is cooperativity
the binding of a ligand at one site affects binding of Ligands at another site
If Kd>>1, it favors
[P][L]
in P+L >< PL
If Kd<<1, it favors
[PL]
in P+L >< PL
How does hemoglobin bind an n, number of O2 molecules in a cooperative fashion?
using positive cooperativity
Hb +nO2 yields Hb(O2)n
Hb binding O2 at one site increases affinity for O2 at another-- cooperative process
Define Y, fractional saturation of Hemoglobin
Y= pO2^4/ pO2^4 +Kd
or Y= [O2]^4/ [O2]^4 +Kd
At n=1, hemoglobin does not show
positive cooperativity
Y= pO2^n/ pO2^n +Kd
n=1-4 lines are averaged into a sugmoidal curve
Define Hill Equation
used for cooperative ligand binding
Hb(O2)n >< Hb +nO2
Y= pO2^n / pO2^n + p50^n
n is the Hill constant/coefficient
Describe differences in the Hill Constant
n=1, non cooperative binding, hyperbolic
n>1, positive cooperativity- sigmoidal
n<1, negative cooperativity- sigmoidal
Hill Equation and Plot FULL
log (y'/1-y') = nlogpO2 - nlogp50
When y (log y'/1-y') is plotted against x (log pO2)
Region of max slope is n
logp50= x intercept
When y (log y'/1-y') is 0
then the y' is 0.5
nlogpO2 = nlogp50 or pO2 = p50
max slope is n
Myoglobin hill plot slope is...
1 because only 1 binding site and is non cooperative
p50= x intercept
oxygen transport torrs
158 inspired air
100 aveolar air
90 arterial blood
capillary 40
interstitial 30
cytosol 10
Cooperativity is designed to...
release O2 at low pressures
weakest afffinity at n=1 and strongest affinity at n=4
Structure of hemoglobin more detailed
1. alpha2beta2 tetramer
2. quaternary structure is dimer of alphabeta dimers
3. alphabeta dimers associate bia hydrophobic/ ionic interactions and hydrogen bonds (not affected by pH)
4.The dimer of alphabeta dimers (tetramer) only has ionic interactions a
Describe conformational changes caused by oxygen binding
Without O2 bound, Fe II is out of heme plane, with O2, is in heme plane, pulling His F8 along with it.
The F helix moves which acts as a trigger that allows for a conformational change creating greater affinity for O2 at other binding sites in hemoglobin.
Cooperativity of binding multiple O2 molecules in hemoglobin results from a...
structural change!
Describe two stable states of hemoglobin
R state: the state oxygen loves and clings to the most
T state: predominates in the absence of oxygen, but oxygen can bind to it...
T state is deoxyhemoglobin
R state is oxyhemoglobin
When deoxy-hemoglobin crystals are exposed to oxygen... they
shatter! proof of structural change
What happens when oxygen binds to hemoglobin in terms of degrees?
one alphabeta pair moves relative to the other by 15 degrees--induced by a movement of Fe II by 0.029 nm
T-state (deoxy Hb) is stabilized by a series of______ that lie at the intersection between the two alphabeta protomers
?
The structural shift from T state to R state predominantly affects what?
the quaternary structure of the alphabeta protomers
Detail the T to R conformation shift
1. His F8 moves with Fe2+ toward heme
2. His F8 packed away in hydrophobic pocket reorients Helix F when moved
3. Helix F moves, producing quaternary shift in the alpha1C-Beta2FG contacts
4. alpha1-beta1 and alpha2-beta2 interactions remain unchanged, so
pO2 for venous blood
30 torr
pO2 for arterial blood
100 torr
p50 for Myoglobin
2.8 torr- no cooperativity
p50 for Hemoglobin
Average is 26 torr- shows cooperativity
1st binding p50 is 30 torr
2/3rd binding p50 is ~12 torr
4th binding p50 is 0.3 torr
T to R transition in hemoglobin must occur simultaneously at...
the interface between the two alphabeta protomers
Describe 4 binding site steps in hemoglobin
1. First binding, low affinity T, no cooperativity
2. Second binding induces T to R, interface destabilized enough for T to R to transition
3. 3rd Binding easier- High Affinity for R state
4. Last binding occurs readily- Very high Affinity for R state- no
Conclusions about Cooperativiy and the Quaternary shift (3)
1. Binding of one O2 results in strain on T-state that tears away C-term ion pairs
2. quaternary shifts to R state increasing binding site's love (affinity) for O2
3. O2 binding become more efficient
Oxygenation causes the F helix of one alpha beta dimer to...
shift relative to the C-term of the opposite alphabeta dimer
Interactions in the structure of hemoglobin
1. dimer of dimers (alphabeta) makes up a tetramer
2. the alpha beat dimer is stable due to hydrophobic interactions at dimer interface working with ionic interactions and hydrogen bonds
3. the alpha1beta1 dimer alpha2beta2 dimer interface forming the tet
4 concepts of quaternary shift and O2 binding
1. T state (deoxyHb) stabilized by ionic interactions
2. Quaternary shift is the movement of residues in the alpha1beta1 dimer- alpha2beta2 dimer interface
3. R state (oxyHb)- possible due to energy harnessed from Fe2+ bond with O2 during 1st O2 binding
4
Describe changes in ionic interactions during T-R transition for T-state
The formation of ion pairs increases the pK values of the group.
Increase pK, decrease acidity (increase pH). Therefore will cling to, and hold hydrogens. MORE BASIC
Describe changes in ionic interactions during T-R transition for R-state
interaction of ion pairs are absent causing a decrease in pK (increasing acidity, lowering pH). Will readily release, throw away protons. Donate protons
Hb releases ____ ____ for each O2 it binds
0.6 H+
Effectors on Hemoglobin's affinity for O2
[O2]
[H+] - Bohr Effect
[CO2]
[2,3 Bisphophoglycerate] - BPG
How does an increase in the [H+] (more acidic, lower pH) affect the binding curve of Hemoglobin for O2?
it moves the Hb binding curve to the right, with a higher p50 value (lower affinity for O2)
For each O2 that binds to hemoglobin, what happens to the environment?
hemoglobin releases ~0.6 H+ and pH decreases (increases acidity)
Decrease in pH (increase in H+) does what to Hemoglobin's affinity?
Decreases it
Increase in pH (decrease in H+) does what to Hemoglobin's affinity?
Increases affinity (more basic)
Christian Bohr
observed the decrease in O2 binding affinity of hemoglobin in response to a decrease in pH
Bohr Effect
Bohr Effect
has important physiological functions in transporting O2 from lungs to respiring tissue
related to blood buffering system- important because a constant biological pH must be maintained for physiological reactions
Blood buffering system
Carbonic Acid (H2CO3) / bicarnonate (HCO3-)
not the only buffer
CO2 (g) >< CO2 (aq)
CO2 (aq) + H2O (l) >< H2CO3 (aq)
H2CO3 (aq) >< H+ (aq) + HCO3- (aq)
----------------------------------------------------
Total Equation: CO2 (g) + H2O (l) >< H+ (aq) + HCO
What happens to the blood buffering system if pH decreases?
If pH decreases, more acidic, so there is an increase in [H+] from metabolic reactions
Equilibrium shifts towards H2CO3
H+(aq) + HCO3- (aq) yields H2CO3 (aq)
and
H2CO3 loses water to produce CO2, expelled by lungs
H2CO3 (aq) yields CO2 (aq) + H2O (aq)
CO2
What happens to the blood buffering system if pH increases?
becomes more basic so there is a decrease in [H+]. Breathing adjusts to introduce more CO2 into blood from lungs to allow ultimate conversion of H2CO3
CO2 (g) yields CO2 (aq)
CO2 (aq) + H2O (aq) yields H2CO3 (aq)
What enzyme accelerates the diffusion of CO2 from respiring tissue back to the lungs?
carbonic anhydrase in red blood cels
What does hemoglobin do with H+ in the T-state
Binds to it.
What does hemoglobin do with H+ in the R state?
Releases it
How does increase pO2 affect uptake/ release of H+ for hemoglobin?
Increase pO2, H+ from Hb is taken by HCO3- to produce H2CO3 yields H2O + CO2
How does decreasing pO2 affect uptake/release of H+ for hemoglobin?
Decrease pO2, H+ from H2CO3 is taken by Hb to produce HCO3-
Show the raw equation for CO2 being expelled in lungs, and HCO3- in tissues preparing to travel back to the lungs
CO2 +H2O >< H+ + HCO3-
What is carbamation
Hemoglobin acts as a CO2 transporter
How is CO2 moved about the body?
1. as HCO3- in blood
2. binds to N-terminal amino acid groups of hemoglobin subunits to form carbamates
carbamates
CO2 bound to N-terminal amino acid groups of hemoglobin subunits
Which of the 2 states of Hb binds CO2 better?
T state
remember, H+ is released upon binding.
Why does the R form shift to T form and release O2?
because T form binds CO2 better, and H+ is released upon binding
CO2 binds to N terminus of Hb liberating a H+ creating the Bohr effect with decreases Hb's affinity for O2 (decrease in pH, decrease in affinity)
Affects of Carbamation
1. aids in transport of CO2 TO the lungs
2. increases Bohr effect by releasing H+ when the CO2 binds to the N terminus of amino acids in Hb
3. Carbamates can form ion pairs stabilizing T state (deoxy Hb)
How much does Hemoglobin aid the transportation of CO2?
~50% transported by the blood for exhaling out of the lungs by hemoglobin
Which state does 2,3-Bisphosphoglycerate (BPG) bind in hemoglobin? How does it effect the binding affinity for O2?
T-state (deoxy Hb)
Decreases affinity for O2
Decribe oxygen saturation of hemoglobin in artieries and veins
Arteries (have a pO2 ~100 torr) allow Hb to become 95% saturated with O2
Veins (have a pO2 ~30 torr) allow Hb to be 55% saturated with O2
How does the presence of 2,3- Biphosphoglycerate (BPG) help Hemoglobin?
Presence of BPG helps Hb unload its O2 into tissues
How does BPG help Hb release O2 from its binding sites?
BPG in T-state (deoxyHb) binds at central cavity which narrows during the T to R quaternary shift releasing the BPG
What does fetal hemoglobin have small amounts if none of?
BPG, it does not readily allow 2,3-biphosphoglycerate to bind
Important for fetus to obtain O2 from mom's circulatory system
Joseph Barcroft
Observed highly purified, stripped Hemoglobin binds O2 with a higher affinity than Hb in whole blood due to allosteric effects. CO2 and BPG are both negative alloseteric effects that diminish hemoglobin's affinity for O2.
Allostery
the binding of a ligand at one site affects the binding of ANOTHER LIGAND at another site
Explain CO2 and BPG affects on binding affinity for O2 in hemoglobin.
CO2 and BPG bind at sites other than O2 binding sites and decrease affinity for O2
Explain Symmetry model of allostery
Hb is capable of existing in T or R state.
Ligands can bind subunit in either conformation and only the conformational change alters affinity for a ligand. The conformational change when all subunits are the same conformation.
Problems:
1. conformational
Explain Sequential model of Allostery
Ligand binding induces conformational change in the subunit it binds and cooperative interactions result
Cooperative Interactions change neighboring subunits that influence its affinity for ligand
Allostery models of O2 binding in Hemoglobin realistically involves
both symmetry and sequential.
Change in T to R states is concerted-symmetry model
Ligand binding in T-state results in small tertiary structural changes that cooperatively induce Hb's final quaternary shift- sequential model
Hemolytic anemia
Mutation in Hb
deficiency in red blood cells, compromises O2 delivery to tissue
Cyanosis
Mutation in Hb. Methemoglobin predominates from mutation (Fe II to Fe III) reducing O2 binding cooperativity, bluish skin results
Polycythemia
Mutation in Hb
increases O2 affinity for Hb- increased red blood cells to compensate for less O2 being released
ruddy complexion
Sickle-cell anemia
Mutations in Hb
single amino acid substitutions
carried by 25% african americans, Hb S
heterozygous suffer mildly
homozygous get sick- Mis-shaped red blood cells can't pass through capillaries
Why are african americans prone to sickle cell amenia genes?
Hb S protects against malaria- heterzygous gets protection
Adaptive advantage like Cystic Fibrosis in europeans making them more resistent to cholera