CHEM 237: Hemoglobin & Myoglobin

Main Ideas of Hemoglobin & Myoglobin

1. Protein Ligand Interactions

2. Oxygen binding properties

3. Structure correlation of Mb and Hb

4. Hemoglobin mutations

Are protein ligand interactions reversible or irreversible?

reversible

What is the molecule that binds called?

ligand

What are ligands typically? and in the form of what?

small, in relativity (ex. amino acid, steroid hormone, small peptide, large growth hormone)

What is the region where the ligand binds on the protein called?

binding site

What force allows ligands to bind?

non-covalent forces (weak forces between molecules or atoms that don't involve chemical bonds)

What are examples of non-covalent bonds seen in ligand bonding?

hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions

Are ligand binding interactions transient or permanent?

transient (temporary)

What state are ligands in a thermodynamically favourable state (lower energy)?

bound ligand = more favorable thermodynamic state

free in solution ligand = less favorable thermodynamic state

What is Ka?

the equilibrium constant of protein and ligand association

How does Protein Ligand affect Ka?

high protein ligand = high Ka

Is the rate of Ka = Kd?

yes

the rate of association (binding) is equal to the rate of dissociation (release), allowing for continuous dynamic exchange

What does a high Ka value mean?

tight binding between protein and ligand

What is the formula for Ka?

Ka = [PL] / [P][L]

What is Kd?

equilibrium constant of dissociation

What is the formula for Kd?

Kd = [P][L] / [PL]

Kd = 1/Ka

What is the relation between Ka and Kd?

they are inversely related to each other

What does a high Kd value mean?

loose binding between protein and ligand

What is an example of ligand and protein binding and dissociation?

affinity chromatography

What is the Fraction of Occupied Binding Sites in a Protein?

the proportion of a protein's available binding sites that are currently occupied by a ligand molecule

how much of the protein is "saturated" with the ligand

What is the symbol used to represent the Fraction of Occupied Binding Sites in a Protein?

θ (theta) or Y

What is the formula(s) to represent the Fraction of Occupied Binding Sites in a Protein?

θ = [PL] / [PL]+[P]

θ = [L] / [L]+Kd

[L] = free ligand concentration

How would you describe the binding curve of the concentration of ligand & fraction bound? Why?

hyperbolic

As ligands are added, it binds to high levels of proteins, therefore the number of available proteins are decreasing and eventually reach a plateau.

When ligand concentration is LOW, what is the fraction bound?

HIGH

When ligand concentration is HIGH, what is the fraction bound?

LOW

What does it mean when [L] = Kd?

[L] = half protein IS bound to ligand and other half is NOT bound to ligand

exactly 50% of the binding sites are occupied and 50% remain unoccupied

What can you solve for once Ka is found?

∆Gº= -RTlnKa

How can you solve ∆Gº in terms of Kd?

∆Gº=RTlnKd

(no negative: -RTlnKa)

What 2 factors does the fraction of bound sites depend on?

[L] = free ligand concentration

Kd= dissociation equilibrium constant

What is the independent variable in a typical binding experiment?

ligand concentration

How can Kd be determined?

graphically or via least-squares regression

What does Kd describe the state of?

the concentration of ligand [L] at which half of the binding sites are occupied

What variable calculates for interaction (binding)?

free energy (∆Gº)

What value of Kd represents STRONG binding?

Kd < 10 nM

What value of Kd represents WEAK binding?

Kd > 10 µM

What protein has a high affinity/strength (low Kd constant)?

avidin (1x10^-15)

this interaction is so tight it can be considered to be irreversible

What protein has a low affinity/strength (high Kd constant)?

calmodulin (3x10^-6)

What is the most efficient way to convert food into energy?

oxidative metabolism

What does Oxidative Metabolism allow for?

diffusion of oxygen through tissues

What does active cells produce that must be removed from the tissues?

CO2

What are the 2 main Oxygen binding proteins?

Hemoglobin and Myoglobin

What does each subunit of Hb and Mb contain?

1 iron containing heme group

How many alpha helices does the Alpha Subunit have? Beta Subunit?

Alpha = 7 alpha helices

Beta = 8 alpha helices

What polymerization is Myoglobin?

monomer

What polymerization is Hemoglobin?

tetramer

Is there a covalent attachment between the heme and protein (Hb or Mb)?

no

What kind of tissue is Myoglobin found in?

muscle

What protein structure folding is Myoglobin?

tertiary

How many peptides is myoglobin made up of?

1 (single polypeptide)

What is Myoglobin responsible for?

storing oxygen and transporting it within muscle tissue

What does Myoglobin make more effective/increase?

the solubility of O2 in cells

&

diffusion rate within the tissue

Where is Hemoglobin located?

in RBC

What protein structure folding is Hemoglobin

quaternary

What is Hemoglobin responsible for?

transports oxygen to tissues from lungs

transports carbon dioxide from tissue to lungs

What does the Heme group fit between?

a hydrophobic pocket (between the C, F, and E helices)

What allows Heme groups to have their absorbance measured?

aromatic strucuture

What is polarity is Heme?

non-polar (due to the structure of its porphyrin ring; typically located in the interior of the protein where it is shielded from water)

How to measure oxygen binding of Heme group?

UV-Vis Spectrophotometry

What about the heme group allows it to absorb light in the UV and Visible range?

it is a strong chromophore (contains a system of conjugated double bonds within its porphyrin ring structure)

Changes in what will change the absorbance of hemoglobin?

changes in iron (Fe) electronic state will change the absorbance

What does Myoglobin's Oxygen Binding Curve look like?

Hyperbolic

What does Hemoglobin's Oxygen Binding Curve look like?

Sigmoidal

What is the key factors that exhibit the relationship of Oxygen binding curve in Myoglobin?

partial pressure of Oxygen (X-axis) & the degree of saturation (θ, Y-axis)

How would you calculate the Degree of Saturation (θ) of Oxygen Binding?

θ = P(O2) / P(O2)+Kd

What is Oxygen Partial Pressure?

the amount of Oxygen dissolved in solution inside the cell or in the blood

What is the value for Partial Pressure of Oxygen?

20% (0.2)

How will oxygen vary in different pressures?

affinity changes

How will oxygen act under LOW pO2?

hemoglobin releases oxygen

How will oxygen act under HIGH pO2?

hemoglobin binds oxygen efficiently

What can we say about the relationship between pO2 and Kd when θ(O2) = 0.5?

pO2 = Kd

What are the 4 functions of Myoglobin?

1. Increases Oxygen solubility in water

2. Increases Oxygen diffusion via MbO2

3. Store Oxygen

4. Not essential for survival

Is Myoglobin lousy or good transport protein?

lousy (bad)

What kind of binding of Oxygen do Lungs need? Tissues? (tight/loose)

Lungs: tight oxygen binding

Tissues: loose oxygen binding

What allows oxygen to bind at different "tightness"?

Cooperativity

a protein with multiple binding sites

(these binding sites must be able to interact with each other)

What's Positive Cooperativity?

when the binding of a ligand to one site increases the affinity of the ligand to bind to another site

What does Hemoglobin transport to tissues?

oxygen (lungs > tissues), carbon dioxide (tissues > lungs)

What concept explains why hemoglobins graph is sigmoidal?

cooperative binding

How does hemoglobin exhibit an example of Positive Cooperative Binding?

as one oxygen molecule binds to a hemoglobin subunit, it induces a conformational change that increases the affinity of the other subunits for oxygen, leading to a rapid increase in oxygen binding once the first few molecules are bound

What does the "S" shape of hemoglobin's oxygen binding curve represent?

the more oxygen is bound, the easier it is for more oxygen to bind further (causing a steep middle section on the curve_

What's the T-state of Hemoglobin?

"tense" state = low affinity for oxygen

binds oxygen poorly at all 4 subunits

What's the R-state of Hemoglobin?

"resting" state = high affinity for oxygen

binds oxygen well at all 4 subunits

What determines whether or not hemoglobin is in the T-state or R-state?

equilibrium

T-state = oxygen levels are low

R-state = oxygen levels are high

Why is the alternation between high affinity and low affinity states a good thing?

allows hemoglobin to efficiently pick up, transport, and release oxygen where needed

How does the T-state and R-state differ in structure?

T-state: constrained and tightly packed structure with lower affinity for a ligand,

R-state: more open conformation with higher affinity for a ligand

Which part of the T-state is the most difficult when binding oxygen?

binding the first 1 or 2 oxygen

When does T-state convert to R-state?

after binding to 2 oxygens

What's an effector molecule?

a molecule/ion that binds to a protien/enzyme to change its activity

What's a Positive Effector?

increase affinity/action

What's a Negative Effector?

decrease affinity/action

What's a Homotropic Effector?

binds to the same site as the substrate

What's a Hetertropic Effector?

binds to a different site than the active site

What environmental condition affects the affinity of oxygen?

blood acidity

What two factors stabilize the T state?

H+ and CO2 (therefore decreases affinity for O2)

Where does CO2 bind to on hemoglobin?

N-terminus of globin (not heme)

How does an increase in CO2 affect Oxygen affinity and pH?

decrease oxygen affinity

decrease pH (more H+)

The presence of what small molecule in the blood affects oxygen affinity?

BPG; stabilizes T state, decrease in affinity for oxygen (increase oxygen in tissue)

What's the Bohr Effect?

the effect of pH on the oxygen dissociation of hemoglobin

decrease in pH (more acidic environment) leads to a decreased oxygen affinity, causing hemoglobin to release more oxygen to tissues; this phenomenon is known as the "Bohr effect"

What does the Bohr Effect state about the curve when acidity increases (pH decreases)?

the affinity of hemoglobin for oxygen decreases

the Fraction of Occupied Binding Sites in a Protein increases (more available sites)

increasing acidity shifts the hemoglobin binding curve away from the Y-axis

What 2 factors make blood acidic when metabolically active?

CO2 and ATP (produce H+)

Will acidic blood (low pH) increase or decrease Oxygen's ability to bind to hemoglobin?

decrease oxygen affinity

in the presence of H+, hemoglobin will prefer to be in it's protonated form than bind to oxygen