ITC (Isothermal Titration Calorimetry)

Report

A single experiment sufficient to obtain all of
the thermodynamic components
 Stoichiometry of the interaction (n)
 Association constant (Ka) & Dissociation constant




(Kd)
Enthalpy (ΔHb)
Free energy (ΔGb)
Entropy (ΔSb)
Heat capacity of binding (ΔCp)


A system is defined as the matter within a
defined region of space (i.e., reactants,
products, solvent)
The surroundings is the matter in the rest of
the universe


The total kinetic energy due to the motion of
molecules (translational, rotational,
vibrational) + the total potential energy
associated with the vibrational and electric
energy of atoms within molecules or crystals.
ΔU= W + Q
ΔU= W + Q
QP = ΔU – W
QP = ΔU – P(V2-V1)
QP = ΔU – P(ΔV)
QP = ΔH
The enthalpy is the heat absorbed or emitted
by a system at constant pressure



Exothermic reaction
ΔH is negative
Emits heat



Endothermic reaction
ΔH is positive
Absorbs heat
 Reaction continues
 Temperature change
occurs
 Power supply is given
to maintain a constant
temperature
difference between the
reaction cell and the
reference cell
 Power supply is
measured

Exothermic reaction:
 Emit heat
 Negative peak on ITV

Endothermic reaction:
 Absorb heat
 Positive peak on ITC

Quantitative technique that can directly
measure:
 the binding affinity (Ka)
 enthalpy changes (ΔH)
 binding stoichiometry (n) of the interaction between
two or more molecules in solution

Gibbs energy changes (ΔG), and entropy
changes (ΔS), can be determined using the
relationship:
 ΔG = -RTlnKa = ΔH-TΔS (where R is the gas constant
and T is the absolute temperature).
As promised…

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
The Energy is conserved
The total energy of a system and its
surroundings is constant
In any physical or chemical change, the total
amount of energy in the universe remains
constant, although the form of the energy
may change.
ΔU= W + Q
 ΔE represents the change in the energy
 Q the heat absorbed by the system
 W the work done on the system
QP = ΔU – W
QP = ΔU – P(V2-V1)
QP = ΔU – P(ΔV)
QP = ΔH
The enthalpy is the heat absorbed or emitted by a
system at constant pressure

The total entropy of a system and its
surroundings always increases for a
spontaneous process
ΔStotal = Δ Ssystem + Δ Ssurroundings
Δ Ssurroundings = - Δ Hsystem/T
Δ Stotal = Δ Ssystem - Δ Hsystem/T
-T Δ Stotal = Δ Hsystem - T Δ Ssystem
Δ G = Δ Hsystem - T Δ Ssystem
 ΔG<0 spontaneous change
 ΔG=0 equilibrium
For a reaction to be spontaneous, the entropy
of the universe, ΔStotal, must increase:
Δ Ssystem > Δ Hsystem/T or
Δ G = Δ Hsystem – T Δ Ssystem < 0
 The free energy must be negative for a
reaction to be spontaneous!


Reactant  Product

Association constant:

ΔG = –RT lnKeq
 Keq = 10–ΔG/1.36
 PL 
G  G  RT ln

 PL 
0
at steady state, at which ΔG=0
 PL 
G   RT ln

 PL 
0
 1
G   RT lnK eq    RT ln
 Kd
 H 0  1  S 0
  
lnK d   
R
 R  T 
0

  RT lnK d 

ADVANTAGES



Immobilization or labeling
필요 없다.
Kd, ΔH can be measured
Can be applied to different
reaction temperature and
pH
DISADVANTAGE
Enormous amounts of
binding partner
◊ Only medium affinity
◊ Limitation for membrane
proteins
◊ High price
◊

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