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Biochemistry 410

Myoglobin and hemoglobin

Vikas Kumar

[email protected]

Bio-Bio Room 321

Office hours 2pm-4pmTue-wed & Fri

mailto:[email protected]

mailto:[email protected]



Overview

• Myoglobin and Hemoglobin

• Structure

• Oxygen binding

• Cooperativity

• Allosteric effects

• Problems



Myoglobin and Hemoglobin

• Most studied proteins in nature

• Both works as a oxygen carrier

• Myoglobin works as a oxygen storage and transport

protein in muscles

• Hemoglobin binds oxygen and transporting it throughout

the body through blood vessels

• Both are globular proteins consisting of heme as a

prosthetic group



Structure

Myoglobin

Monomer

153 a.a

17.2kDa

Hemoglobin

Tetramer

α2β2

64.5kDa

Iron prefers to interact with 6 ligands

4 porphyrin N atoms

N from His F8

O2 is 6th

Heme



Conformational change after O2 binding

• Without O2, Fe2+ is out of heme plane

• O2 pulls the Fe2+ into the heme plane

• Fe2+ pulls its His F8 ligand along with it

•This change means little to Mb, but lots

to Hb!



Ligand BindingLigand = O2

We know

pO2

Y =

Kd + pO2



The “affinity” constant, p50

50% of myoglobin is saturated with O2

pO2

Y =

p50 + pO2

• For Mb partial pressure of 2.8 torr is sufficient for half saturation



Cooperative ligand binding in Hb

pO2n

Y =

p50 + pO2n

Hills

equation

The binding of O2 to Hb is cooperative – binding ofoxygen to the first subunit makes binding to the othersubunits more favorable.

p50=26 torr



Hill Plot



Allosteric effectors

• Oxygen is an allosteric effector as well as a ligand

• Binding of O2 to Hb is affected by several agents, including

• H+

• CO2

• 2,3-bisphosphoglycerate

• The effect of H+ is particularly

important. This is the Bohr effect.

• Deoxy-Hb has a higher affinity for

H+ than oxy-Hb.

• Thus, as pH decreases, dissociation

of O2 from hemoglobin is enhanced



Allosteric effectors

CO2 has two effects:

1. CO2 diminishes O2 binding by decreasing pH.

• HCO3- is transported with the blood

back to lungs

• Hb binds O2 in the lungs releasing H+

• H+ reacts with HCO3- and CO2 is

released

2. Hb also acts as CO2 transportor

2,3-Bisphosphoglycerate as allosteric effector:

• Found in RBC’s

• Binds preferentially to deoxy-Hb form

• Binds in the central cavity of tetramer

• Negative charge interacts with 2Lys, 4His and

2N-termini



ProblemA protein has binding affinity for its ligand (a peptide) of Ka = 2 105 M at

pH 5.0 and 25oC. At what concentration of the ligand is half of the protein

bound?



ProblemWhat fraction of the Hb is bound at partial pressure of 1.25 torr (p50 = 5

torr)?

At what oxygen concentration will be 80% of the Hb bound?



ProblemWhat conclusions about possible cooperativity of the binding can you

draw from these plots? What does different shape of the binding curves

tells you about the affinity of the ligand?