Hemoglobin and Myoglobin

do ligands bond via covalent or non-covalent forces?

non covalent

what does “binding reversibly mean”

binding is not permanent

define the terms equilibrium composition, equilibrium, and equilibrium constant

equilibrium composition —> amount of P, L and PL in a system once it has reached equilibrium

equilibrium —> rate of binding = rate of unbinding

equilibrium constant —> how strongly a ligand binds to a protein

define the assocation and dissocation constant

association

• ka → how strongly a ligand (oxygen) binds to a protein

  • large ka - strong

  • small ka - weak

dissociation

• kd → how easily a ligand (oxygen) unbinds from a protein

  • large kd → easily comes off

  • small kd → tightly bound

what does theta tell us

the fraction of occupied binding sites on a protein

theta depends on what 2 things:

1. free ligand concentration

2. kd = how strongly a ligand binds to the protein

what does kd = lignad concentration mean

occurs at theta = 0.5

• represents the point where half the binding sites are occupied and the other half are free

what is oxidative metabolism

using oxygen to convert nutrients (eg. glucose) into energy (ATP)

list 2 oxygen binding proteins found in our body. define what an oxygen binding protein is

oxygen binding protein → protein that help transport or store oxygen in the body

hemoglobin and myoglobin

in hb, how many alpha helices are in the alpha and beta subunit

alpha - 7

beta - 8

how many subunits are in myoglobin and hemoglobin

myo - monomer (1)

hemoglobin - tetramer (4)

which 3 helices form the hydrophobic pocket in myoglobin and hemoglobin

c, f, and e helices

what atom in a heme group binds to oxygen

iron

what degree of protein structure is hemo and myo

hemo - quaternary (contains multiple subunits)

myo - tertiary at most (only one subunit)

T/F Disulfide bonds can attach a heme to its protein

False

there are no covalent attachments between the heme and protein

where do you find myoglobin & hemoglobin

myo - muscle tissue

hemo - red blood cells

what is the function of myoglobin and hemoglobin

myo - store and transport o2 within muscle tissue

hemo - transport o2 from the lungs to tissue

what are the subunits that make up hemoglobin

tetramer (4)

• two alpha subunits and two beta subunits

what is responsible for the red colour in muscle tissue

myoglobin

what happens if oxygen binds to the iron (fe2+) in the heme group straight on?

it would oxidize fe2+ → fe3+

• fe3 would not be able to bind to o2 effectively

• no function

what is the importance of Histidine when oxygen binds to iron in the heme group?

iron is attached to His

• when o2 binds to iron, iron moves slightly, pulling on His

• Since His and Fe are attached, His also slightly moves, triggering a larger change in hemoglobin structure

does heme absorb more or less light when bound to oxygen

absorbs less light

what does uv-vis spectrophotometry help us determine

determine the portion of Hb that is oxygen bound or oxygen free

• dete

electronic state meaning

different energy levels that electrons can occupy in an atom

what does it mean if we say myoglobin is easily saturated?

saturated refers to how much myoglobin in a population is bound to oxygen

• saturated myoglobin means myoglobin is bound to o2

• myoglobin can easily bind to o2, therefore it gets saturated easily

what does it mean when we say the myoglobin binding curve is hyperbolic

hyperbolic → graph rises quickly and then levels off

• rises quickly bc myoglobin binds to o2 easily and in a high o2 environment, it gets saturated easily

• eventually levels off bc theres no more binding sites to bind onto o2

why do we use partial pressure to measure how much oxygen is in an environment instead of concentration?

concentration would refer to the amount O2 in a volume of solution

partial pressure refers to the pressure exerted by o2 molecules in a mixture of gas

• o2 is gas

what is the definition of the partial pressure of o2

a measure of the amount of o2 dissolved in an aqueous solution inside cells or blood

• aka how much o2 is available

what does degree of saturation mean

how much hb/mb is bound to o2

• how many binding sites are available on hb/mb to bind to o2

what does mb do in a low and high pO2 environment?

mb has a strong affinity for o2

low po2 - keep it tightly and not release

high po2 - pick up as much as possible

oxygen is soluble/insoluble in water

insoluble

what increases solubility of o2 in water

binding to mb

T/F Oxygen is a small gas molecule and can easily diffuse passively through cells

False

despite being small, it has terrible diffusion. therefore its terrible at moving and has trouble spreading out

why do aquariums have pumps to swirl water & why are lakes oxygen poor

oxygen is terrible at diffusion and doesn’t spread easily/travel well

• aquariums have pumps to spread oxygen around, so that fish can get enough oxygen to breathe

• lakes have no movement and are oxygen-poor bc oxygen can’t spread evenly

  • harder for organisms to survive in lakes

efficient oxygen transport:

• _______ (tight/low) of O2 in high pO2 environments

• _______ (tight/low) of O2 in low pO2 environments

tight;weak

what is positive cooperativity

the first binding event that increases the affinity for the other binding sites

T/F Myoglobin can’t transport O2

F

it is able to, but it is weak for oxygen exchange in our body since its affinity to O2 is too high

• needs the help of Hb

how does teh sigmoidal binding curve of Hb reflect cooperative binding

because binding one o2 molecule makes it easier to bind even more O2

• this is shown in teh graph that gradually increases

explain why Hb is such an amazing transport protein by explaining what happens at low and high pO2 environments.

high pO2 - will pick up a lot of o2 easily

low pO2 - unlike Mb, it will release o2 more readily at environments w low oxgen

T/F Binding of O2 to a binding site on hb is completely random

T

T/F When Hb is bound to an O2, it can still switch between states

T

depends entirely on the environment (high or low o2)

Q: What would happen if Hb only had a high affinity for O2?

It would be just like Mb. It would only hold onto O2 and never release it, making it a terrible protein for transporting oxygen. 

• Being able to switch between states is what makes it a good transporter!

4 o2 bind onto hb. what happens to hb structurally between the first O2 binding and then the last two

when the first o2 bind to hb, it is in its T state

• structurally very difficult for O2 to access binding sites

afterwards, Hb undergoes a structural change from T to R

• the structure of Hb in the R state makes it much easier for the last O2 to bind

T/F No oxygen can bind to Hb in its T state

F

Binding is possible, it is just very difficult, compared to Hb in its R state

how is O2 both the substrate and homotropic effector

When one O2 binds, it makes it easier for another O2 to bind

difference between homotropic and heterotropic effector?

homotropic - binds to the same site as the substrate

heterotropic - binds at a site other than the active site

what would a leftward and rightward shift of the Hb binding curve mean

rightward shift —> lowers oxygen affinity

leftward shift → increases oxygen affinity

what effect does increasing acidity have on Hb

lowers its oxygen affinity bc acidity stabilizes the T-state

• oxygen is more likely to be released.

how does metabolism affect hb binding affinity

metabolism includes breaking down ATP and releasing CO2 and H+ into the bloodstream, which makes the blood more acidic

• bohr’s effect: acidic blood stabilizes the t-state of Hb, decreasing its affinity to oxygen, making it easier to release O2.

During T state, His has a ________ (low/high) pKa

During R state, His has a ________ (low/high) pKa

pka is for H+ (bohr proton)

high;low

how does the C terminus of His stabilize the T state of Hb

His bound to H+ carries a positive charge

C terminus of His carries a negative charge

the positive and negative charge forms a salt bridge, which is what stabilizes the T state

T/F Binding of Bohr Proton to Hb causes a structural change

T

fill in the blank with either start/end

bohr protons bind to the ______ of the beta subunit of Hb and CO2 binds to the _____ of the beta subunit

end;start

what is the last amino acid in the beta subunit of hb

His146

how does the formation of carbamate contribute to bohr’s effect

carbamate is formed when CO2 binds onto Hb at the N-terminus on one of the beta sheets

formation of carbamate releases a H+

this H+ binds onto the C-terminus of Hb, stabilizing the T state, lowering O2 affinity

what is the haldane effect. explain the structure & trend

deoxygenated hb carries more CO2 compared to oxygenated Hb.

this is bc hb in the t state has a more open and exposed structure. it particularly exposes the N terminus region.

CO2 binds to an N-terminus, forming carbamate and releasing H+. this causes bohr effect to take place: H+ binds onto the C terminus of Hb to stabilize T-state

blood acidity increases and o2 affinity decreases

what causes Hb to release O2 for cells/tisses?

when salt bridges form due to H+ binding to His in the beta sheets of Hb

• bc it stabilizes the T state

T/F BPG is able to bind to Hb in both its T and R staet

F

only T state. bpg has no effect on hb in the r state.

In the T state, the central cavity in Hb is _______ (smaller/larger)

In the R state, the central cavity in Hb is _______ (smaller/larger)

larger;smaller

what type of charges does BPG have

5 negative charges

why cant BPG bind to Hb in its R state

In its R state, the central pocket of Hb is too small. BPG is not able to bind. 

in t state, the pocket is exposed and bigger

how many bpg molecules can bind to a hemoglobin tetramer

only 1

bc bpg binds to the central pocket of hb

which location of hb does bpg bind to

central pocket

why does BPG increase P50 for Hb?

bpg decreases hb’s affinity for oxygen

p50 refers to the amount of partial pressure of oxygen required to get 50% of hb saturated

• partial pressure → the amount of o2 in the environment

if hb doesn’t want to bind to o2 at all (low affinity), it would require more oxygen in the environment to get more hb to be saturated (bind to oxygen) right

if we’re in a high altitude environment, why would the body produce more bpg?

high altitude - less o2 in environment

bpg decrases hb’s affinity to o2

• the hb bound to o2 will release it, giving it to cells that require o2 to function, since they can’t get any o2 from the environment.

how does BPG affect fetal hb?

it doesnt. fetal hb has a low affinity to BPG, therefore will have a higher affinity to O2

• this is useful to assist in oxygen transfer from the mother to baby, to help with baby’s development & growth

at high altitudes, _______ (more/less) O2 is dropped off in the body

high altitudes - low o2 environment

• we are breathing in less o2

since we’re breathing in less, body compensated by producing more bpg

• bpg production lowers hb’s affinity for o2

• the hb that’s bound to o2 will let go of it, giving it to tissues that need it

what is the protein component of mb called and what is its function

globin

• a large protein structure that stabilizes the heme gorp and enables binding adn release of o2

what is the purpose of the heme group in Mb

stabilizes the tertiary structure of Mb

what is the structure of Mb composed of?

8 a-helical helices, labelled from A to H

• made up of 153 aa

which 2 subunits in Hb have the most interactions

a1 and b1

• 35 contacts

which interface in hb is responsible for switching Hb from the R to T state once it binds to O2

a1b2 - 19 contacts

• important for oxygen binding and conformational changes

hb and mb are very similar in structure, would their sequences be similar?

no. they share 12% of similarities in their sequences

Hb and Mb share a 12% similarities in their sequences, what functions would the aa in that 12% region carry?

the aa in that sequence would be involved in heme binding and orienting oxygen

what is the name for the a1b1 dimer in Hb

basic dimer

describe the shape of the Mb adn Hb binding curve

Mb - hyperbolic

hHb - sigmoidal

what is the bohr effect

increasing acidity in bloodstream will shift the Hb binding curve to the right (lowers affinity for O2)

what aa do bohr protons bind onto

c-terminus histidine residues

what stabilizes the bond between bohr proton and histidine

the carboxyl group

• forms a salt bridge that is stabilizing

which aa does co2 bind onto? which terminus?

N terminus of histidine

how does the central region of BPG affect its binding to Hb? Consider R and T states

BPG can bind to Hb in its T state because the central region is large

In Hb’s R state, the central region is smaller and BPG physically cannot bind on

what is Mb’s cofactor?

Heme

• helps Mb bind onto oxygen

how many tertiary structures make up Mb and Hb?

Mb - one

Hb - 4

how many alpha helices make up the alpha and beta subunit of hemoglobin

alpha subunit - 7 alpha helices

beta subunit - 8 alpha helices

explain the structural changes that occur when O2 binds to Hb

1. O2 binds to Fe2+ in heme, pulling some e- away from fe2+

2. fe2+ becomes smaller and moves towards heme plane (just a bit)

3. small movement will pull on HisF8 thats attached to the F-helix

4. Movement of F-helix disrupts interfaces, leading to a large rotation

5. the rotation weakens sal tbridges and the stabilizing interactions that kept Hb in the T state

which interface in hb contains the bpg binding site

b1b2

which Hb is derived from beta-Hb

gamma (Y)