Hemoglobin 1 - O2 binding & cooperativeity 09/09 Traish Flashcards
function of Mb vs Hb
Mb - O2 storage
Hb - O2 transport
why do we need oxygen?
- oxidative metabolism and energy production -> tissue survival and function
- oxidizing dietary substrates to yield energy & CO2
why do we need O2 carriers? 4 reasons
- diffusion not adequate for large human bodies
- gas exchange through impermeable external skin layer impossible
- O2 has low aqueous solubility and cannot facilitate metabolic needs of body as naked gas
- O2 is strong oxidizing agent and at necessary high concentrations would harmfully oxidize tissues and cells
how many grams of Hb are in 1 L of blood?
150 grams Hb
how much O2 can 1g of Hb bind?
~1.5 ml O2
how much O2 can 1L of blood bind?
~225 mL O2
150 g Hb / L blood
~1.5 ml O2 / g Hb
150 x 1.5 = 225
how much naked O2 dissolves in blood at physiological temperature?
2.3 ml
how much more O2 can travel in blood due to Hb?
~ 90 to 100 times more
~225 ml O2 with Hb / 2.3 ml O2 naked
how much faster would blood have to circulate to deliver adequate O2 if there were no Hb?
~ 90 to 100 times faster
~225 ml O2 with Hb / 2.3 ml O2 naked = <100
~how quickly does blood circulate under resting conditions?
once every 60 seconds
or 18 m/s
what is the atmospheric partial pressure of oxygen
160 mmHg
Hb transport reduces O2 partial pressure from __ to __ as it reaches tissues, which minimizes harmful tissue oxidation
100-160 mmHg to 30-40 mmHg
Hb facilitates __ transport to tissues an __ transport to lungs
O2 to tissues and CO2 to lungs
what are 3 properties of a “preferred oxygen carrier”
- binds O2 at high pO2
- does not oxidize cellular components
- gives up O2 on demand
in what state is iron bound to the prosthetic group in Hb and Mb?
iron (II)
Fe^2+
ferrous
ferrous iron is…
Fe^2+
ferric iron is…
Fe^3+
ferrous to ferric iron transition involves __
ferric to ferrous iron transition involves __
Fe^2+ –> Fe^3+ = oxidation
Fe^3+ –> Fe^2+ = reduction
what kind of bonds hold heme in its crevice in Hb and Mb
non-covalent interactions
is the heme crevice in Hb and Mb hydrophobic or hydrophilic?
hydrophobic - to prevent iron oxidation before O2 binding
T/F heme is a prosthetic group in Hb and Mb
true
how many ligands can ferrous iron usually bind?
6
how does Fe2+ bind protoporphyrin IX?
it bonds to the four N atoms of the porphyrin rings
how many bonds does Fe2+ form with protoporphyrin IX?
4
in a heme group in Hb and Mb, what ligands occupy the 6 possible coordinate bonds in Fe2+?
- 4 occupied by N atoms of porphyrin rings in protoporphyrin IX
- 1 occupied by proximal histidine F8 (8th residue on helix F; his 93 in Mb, his 92 in b-Hb, his 87 in a-Hb)
- 1 occupied by O2 or empty but stabilized by distal hovering histidine E7 (his 64 in Mb, his 63 in b-Hb, his 58 in a-Hb)
the 5th coordination site on Fe2+ is occupied by what?
the proximal histidine F8 (8th residue on helix F; his 93 in Mb, his 92 in b-Hb, his 87 in a-Hb)
the 6th coordination site on Fe2+ is occupied by what?
O2, or empty but stabilized by distal hovering histidine E7 (his 64 in Mb, his 63 in b-Hb, his 58 in a-Hb)
which are the proximal and distal Hb histidines that interact with heme, and why are they so named?
- proximal his F8 (8th residue on helix F; his 93 in Mb, his 92 in b-Hb, his 87 in a-Hb) “proximal” because covalent bond holds it closer to heme than hovering distal E7 his
- distal his E7 (his 64 in Mb, his 63 in b-Hb, his 58 in a-Hb) “distal” because it hovers further away from heme than covalently bonded proximal his F7
what is the oxidation state of iron before O2 binds heme? what is the oxidation state of iron when O2 is released from heme?
ferrous Fe2+ before O2 binding
returns to ferrous Fe2+ after O2 release
in what tissues is Mb primarily located as a storage reserve of O2?
skeletal muscle
Mb accepts O2 from Hb in … and then delivers O2 to …
- accepts O2 from Hb in the circulating blood
- delivers O2 to the mitochondria
binding of O2 to Mb is described by what kind of binding curve?
a hyperbolic binding curve
as pO2 increases, saturation of O2 binding sites is approached asymptotically
what is the equation for the O2 - Mb binding curve?
theta = pO2 / (P50 + pO2) theta = fraction of O2 bound Mb sites pO2 = O2 partial pressure P50 = O2 partial pressure for half saturation
with regard to O2 binding, a low P50 signifies…
high affinity for oxygen (e.g. Mb)
P50 is partial pressure at which half of all bidning sites are saturated with O2
which is lower, P50 of Mb or Hb?
P50 for Mb is lower, must have higher O2 affinity to extract O2 from Hb in blood
how does Mb extract O2 from blood?
P50 for Mb is lower than for Hb, so it has a higher affinity for O2 and will pick up O2 as Hb drops it off at the lower O2 concentrations in tissues (~30-40 mmHg)
how does Mb deliver O2?
at normal O2 concentrations in capillaries, Mb in adjacent tissues is nearly saturated. when cells are metabolically active, their internal pO2 falls to lower levels and Mb releases O2
how many amino acids are there in Mb?
153
how many polypeptide chains make up Mb?
1
T/F the Mb molecule is a very compact globular molecule
true
how many a-helices make up the tertiary structure of Mb?
8 (A-H)
__ a-helices in Mb are terminated by this amino acid residue
4 a-helices in Mb are terminated by proline residues
approximately what percentage of the Mb polypeptide chain is in a-helical structure?
75%
T/F Mb is compact with very little empty space inside
true
Mb is expressed mainly in what tissue?
skeletal muscle
Mb releases O2 only when tissues are __
hypoxic
how does the affinity of myoglobin for O2 change with O2 concentration?
it doesn’t. binding affinity is an intrinsic property of Mb. the molecule does not change (e.g. change conformation) to actively “release” O2, it just becomes less saturated at lower pO2 because oxygenated and de-oxygenated Mb exist at equilibrium with one another i.e. l’chatlier’s principle
does Mb exhibit cooperativity?
no.
T/F Mb can be found in most tissues
false - it is mainly found in tissues with high O2 demand, e.g. skeletal muscle
affinity signifies…
how stongly / how long a substrate is bound
where in Mb does heme bind
in a hydrophobic pocket/crevice
the residue that binds heme in Mb is…
his 93 (F8) the "proximal" histidine
the distal Mb residue that hovers over the O2 binding site in heme is…
his 64 (E7) the "distal" histidine
the six coordinate bond structure of Fe2+ is termed __
octahedral
octahedral coordinate binding structure includes how many bonds?
6 (4 planar, 1 above, 1 below)
the residue that binds heme in beta Hb is…
beta his 92 (F8)
the residue that binds heme in alpha Hb is…
alpha his 87 (F8)
the “proximal histidine” residues that bind heme in Mb and alpha and beta Hb are…
Mb his 93 (F8)
beta his 92 (F8)
alpha his 87 (F8)
the residue that hovers over the O2 binding site of heme in beta Hb is…
beta his 63 (E7)
the residue that hovers over the O2 binding site of heme in alpha Hb is…
alpha his 58 (E7)
the “distal histidine” residues that hover the O2 binding sites of heme in Mb and beta and alpha Hb are…
Mb his 64 (E7)
beta his 63 (E7)
alpha his 58 (E7)
what is the role of E7 in oxy Hb and Mb?
when O2 is bound, his E7 sterically alters O2 binding so that O2 is not bound in the optimal conformation (O2 binds crooked), lowering O2 binding affinity and making easier to remove when needed
what is the role of E7 in deoxy Hb and Mb?
when O2 is not bound, his E7 protects the site from water and prevents Fe2+ oxidation
ferroprotoporphyrin is another name for __
heme
another word for heme is __
ferroprotoporphyrin
heme is the result of the combination of __ and __
ferrous iron and protoporphyrin IX
T/F heme alone is sufficient to protect the iron atom from oxidation
false - O2 will oxidize ferrous iron to ferric iron in the absence of a protein like Hb or Mb
how do Hb and Mb protect heme from irreversible oxidation?
the heme crevice is hydrophobic to exclude water and prevent oxidation by water, and his F7 prevents oxidation by O2 with a degree of steric hindrance
what is the Ka for Mb
Ka = association constant Ka = [MbO2] / [Mb] [O2]
what does Ka refer to
association constant [products] / [reactants]
what does Kd refer to
dissociation constant [reactants] / [products]
association and dissociation constants refer to…
how slow or fast the bound molecule associates or dissociates
how are Ka and Kd related
Ka = 1/Kd Kd = 1/Ka
what does the term “fractional saturation” refer to with respect to Mb
theta = [MbO2] / [Mb] + [MbO
what is the Ka for Mb
Ka = association constant Ka = [MbO2] / [Mb] [O2]
what does Ka refer to
association constant [products] / [reactants]
what does Kd refer to
dissociation constant [reactants] / [products]
what are 3 alternative equations relating fractional saturation of Mb and oxygen concentration
theta = pO2 / (pO2 + P50) -or- theta / (1 - theta) = (pO2 / P50)^nH -or- log [theta / (1 - theta)] = nH log pO2 - nH log P50
nH = hill coefficient (1 for Mb, (~)1-4 for Hb)
on an Mb or Hb binding curve, P50 signifies
the oxygen concentration or (oxygen partial pressure) at which 50% of Mb binding sites are occupied (O2 conc at 50% Mb saturation)
what does the term “fractional saturation” refer to with respect to Mb
theta = [MbO2] / [Mb] + [MbO2]
an oxygen binding plot for Mb and Hb plots…
a Hill plot for Mb and Hb plots…
normal plot: fractional saturation vs pO2
Hill plot: log fract sat / 1-fract sat vs log pO2
what does Ka refer to
association constant [products] / [reactants]
a Hill plot plots this equation…
x-intercept =
slope =
log [theta / (1 - theta)] = nH log pO2 - nH log P50
x-intercept = log P50
slope = nH (nH = 1 for Mb; for Hb (~)1<4)
(near P50 Hb nH~3.5)
association and dissociation constants refer to…
how slow or fast the bound molecule associates or dissociates
how are Ka and Kd related
Ka = 1/Kd Kd = 1/Ka
what does the term “fractional saturation” refer to with respect to Mb
theta = [MbO2] / [Mb] + [MbO2]
fractional sat = ratio of bound Mb / total Mb
what is the Kd for Mb
Kd = [Mb] [O2] / [MbO2]
how is binding affinity related to Kd?
inversely.
Kd down, affinity up
Kd up, affinity down
according to Henry’s Law, the concentration of any gas dissolved in solution is proportional to…
the partial pressure of that gas above the solution
what is the equation relating fractional saturation of Mb and oxygen concentration
theta = pO2 / (pO2 + P50)
on an Mb or Hb binding curve, P50 signifies
the oxygen concentration or (oxygen partial pressure) at which 50% of Mb binding sites are occupied (O2 conc at 50% Mb saturation)
a Hill Plot for Mb and Hb plots…
log fract sat / 1-fract sat vs log pO2
log [theta/(1-theta)] on y axis
log pO2 on x axis
an oxygen binding plot for Mb and Hb plots…
a Hill plot for Mb and Hb plots…
normal plot: fractional saturation vs pO2
Hill plot: log fract sat / 1-fract sat vs log pO2
the relevant equation for a Hill Plot is…
log [theta / (1 - theta)] = nH log pO2 - nH log P50
what do the respective Hill plots for Mb and Hb look like?
-Mb is linear with a slope (nH) of 1 and a lower P50
Hb is sigmoidal with slope (nH) between (~)1 and 4 (<1 at tails at low and high log pO2, about 3.5 at pO2 near P50) and a higher P50 than Mb
T/F the Hill coefficient (nH) indicates the number of subunits participating in binding
false - it indicates “degree of cooperativity”
(a proteins quaternary structure may involve multiple binding subunits but exhibit no cooperativity and an nH of 1, or even “negative cooperativity”)
does a Hill coefficient (nH) value of 1.1 indicate cooperativity?
no - could be due to small experimental error
on a Hill plot, what is the nH for Hb?
between ~1 and 4
at low and high pO2, nH is slightly < 1
at pO2 near P50, nH ~3.5
a Hill coefficient (nH) of 1 indicates…
non-cooperative binding (e.g. Mb)
a Hill coefficient (nH) of >1 indicates…
cooperative binding (e.g. Hb)
Hb is composed of __ subunits
4
2 alpha
2 beta
T/F the Hb tetramer is approximately spherical in shape
true
T/F the alpha subunits in Hb are identical
false - they may or may not be identical (a1 and a2 differ by 1 amino acid and are transcribed from separate genes)
T/F the beta subunits in Hb are identical
true - there is only one b-subunit gene
how many genes code for a-Hb subunits
2 (a1 and a2 differ by 1 amino acid)
how many genes code for b-Hb subunits
1
what do the respective Hill plots for Mb and Hb look like?
-Mb is linear with a slope (nH) of 1 and a lower P50
Hb is sigmoidal with slope (nH) between 1 and 4 (1 at tails at low and high log pO2, about 3.5 at pO2 near P50) and a higher P50 than Mb
why is cooperative binding necessary in Hb?
makes possible to switch from strong binding state at high pO2 in lungs to weak binding state at low pO2 in tissues
T/F Hb participates in maintaining plasma pH
true - by transporting CO2 and H+ to lungs
on a Hill plot, what is the nH for Hb?
between 1 and 4
at low and high pO2, nH ~1
at pO2 near P50, nH ~3.5
how many aa residues in a-Hb subunit?
141
how many aa residues in b-Hb subunit?
146
when subjected to a 2 M urea solution, Hb dissociates into…
a-b dimers
which Hb subunit interactions are stronger, those a-a and b-b or a-b?
a-b interactions are stronger
in Hb, how far apart are heme prosthetic groups from one another?
2.5 nm
T/F the source of cooperative oxygen binding in Hb can be attributed to heme-heme interactions
false - heme groups within the molecule are not close to one another
T/F heme groups are located deep within Mb and Hb molecules
false - heme groups are located close to the surface in relatively superficial hydrophobic clefts in the molecules
what are the 2 conformational states of Hb
oxyHb
deoxyHb
what 4 molecules alter the affinity of Hb to O2?
- O2
- H+
- CO2
- BPG (bisphosphoglycerate)
why is cooperative binding necessary in Hb?
makes possible to switch from strong binding state at high pO2 in lungs to weak binding state at low pO2 in tissues
T/F Hb participates in maintaining plasma pH
true - by transporting CO2 and H+ to lungs
the change in conformation from oxyHb to deoxyHb is called…
an allosteric shift
the Mb binding plot is __
hyperbolic
the Mb Hill plot is __
linear
if pO2 in lung is 100mmHg, pO2 in capillary is 10mmHg, and P50 Hb is 26 mmHg, and nH for Hb is 2.8, how much O2 is delivered to capillaries? how much would be delivered if Hb was non-cooperative?
theta / (1-theta) = (pO2 / P50)^nH
theta lungs = 0.98
theta capillary = 0.063
.98 - .063 = 0.92 = % Hb subs that delivered O2
for non-coop, 0.51 = % Hb subs that delivered O2
the Hb Hill plot is __
sigmoidal
why is a sigmoidal binding curve necessary in Hb?
makes possible to switch from strong binding state at high pO2 in lungs to weak binding state at low pO2 in tissues
what is the reaction equation for O2 binding to Hb
Hb + nO2 Hb(O2)^n
n is a number between 1 and 4
how can you approximate Kd from a Hill plot?
P50 (x-intercept) approximates Kd
the abscissa value on a Hill plot refers to
the x-axis value (the value of log pO2)
is the slope of the Hb binding curve on a Hill plot at extreme (high or low) pO2 equal to 1 or less than 1?
slightly <4) state
what is advantageous about the Hill plot versus a normal binding plot for Mb and Hb?
2 things:
- can visualize transition from non-coop to coop more easily
- gives a direct numerical measure of degree of cooperativity from slope (nH), the Hill coefficient
what does it mean when nH = 4 for Hb?
the molecule is wholly cooperative and only wholly unliganded and wholly liganded molecules exist (hypothetical situation - never happens. max nH ~3.5)
what is the Hill coefficeint (nH) for a hypothetical wholly cooperative situation?
nH = n, the number of subunits
what is the functional difference between Hb and Mb?
cooperativity in Hb. possible because the oxygenation state of one subunit can be communicated to the others
what is the maximum Hill coefficient (nH) for Hb?
~3.5
(4 only in hypothetical situations… would indicate that only fully oxyHb or fully deoxyHb are present, not partially oxyHb present)
if pO2 in lung is 100mmHg, pO2 in capillary is 10mmHg, and P50 Hb is 26 mmHg, and nH for Hb is 2.8, how much O2 is delivered to capillaries?
theta / (1-theta) = (pO2 / P50)^nH
theta lungs = 0.98
theta capillary = 0.063
.98 - .063 = 0.92 = % Hb subunits that delivered O2
