28th Jan 2015 S2004 - Isothermal titration calorimetry
S2004
Methods for characterization of
biomolecular interactions
- classical versus modern
Isothermal Titration Calorimetry (ITC)
Eva Dubská
email: eva.dubska@ceitec.cz
Outline
 Calorimetry - history + a bit theory
 Isothermal titration calorimetry
– Applications
– Instrumentation
– The Raw ITC Data
– Evaluation of ITC Data
 Receptor - Ligand Interactions („thermodynamics behind“)
 ITC data – examples
– „c values“
 Experimental set-up
 Sample preparation
 Troubleshooting
28th Jan 2015 S2004 - Isothermal titration calorimetry
28th Jan 2015 S2004 - Isothermal titration calorimetry
Calorimetry
Calorimetry (Latin calor - heat, Greek metry - to measure) is the
termodynamic technique based on the measurement of heat that may be
generated (exothermic process), consumed (endothermic process) or simply
dissipated by a sample.
A calorimeter is an instrument used for measuring the quantity of heat
absorbed or released in process of a chemical reaction.
1 calorie - express quantity of heat necessary to raise the temperature of 1 g
of water by 1°C.
Heat is generated by almost all processes
(physical, chemical or biological)
28th Jan 2015 S2004 - Isothermal titration calorimetry
Calorimetry - heat changes detection
Lavoisier-Laplace calorimeter
The ice calorimeter was developed in the period 1782 to 1784 by the French scientists Antoine
Lavoisier and Pierre-Simon Laplace. The central space of inner chamber contained burning oil, or
an animal such as a guinea pig. The surrounding chamber contained ice. Heat produced by the
animal can be measured indirectly, by assessing the amount of water that elutes from the bottom
of the chamber, which is the impact of the animal's heat on the ice in the outer chamber.
Antoine-Laurent de Lavoisier (1743-1794)
Inner chamber
snow
ice
Lavoisier medal
 INDIRECT CALORIMETRY - calculates the heat
generated by living organisms when their
metabolic processes yield waste carbon dioxide.
 DIRECT CALORIMETRY - heat generated by living
organisms may also be measured by direct
calorimetry, in which the entire organism is
placed inside the calorimeter for the
measurement.
• different types of direct calorimetry
• sample is placed in the calorimetric cell
Calorimetry
Energy expenditure
from the O2 consumption
28th Jan 2015 S2004 - Isothermal titration calorimetry
28th Jan 2015 S2004 - Isothermal titration calorimetry
Calorimetry
Heat is generated by almost all processes
(physical, chemical or biological)
 heat associated with biological
reactions
 changes in animal metabolism
resulting from nutrition, stress, etc.
 bacterial growth rates in fermenters
 interactions between molecules
 chemical reactions
Microcalorimetry
Differential Scanning Calorimetry
 constant temperature rate
 thermal analysis („titrations“)
 what happend when we heat/cool down the
system?
 During a change in temperature, DSC
measures a heat quantity, which is released
or absorbed excessively by the sample on
the basis of a temperature difference
between the sample and the reference
material.
Isothermal Titration Calorimetry
 constant temperature
 ligand titration
 what happend when two (bio)molecules
interact? (constant temperature)
 Heat is released or absorbed as a result of
the redistribution and formation of noncovalent
bonds when the interacting
molecules go from the free to the bound
state.
VP-DSC
VP-ITC Auto-iTC200 iTC200
28th Jan 2015 S2004 - Isothermal titration calorimetry
Microcalorimetry
Differential Scanning Calorimetry
 Biomolecular stability in solution
 provides insights into mechanisms of unfolding and
refolding
 Midpoint (Tm) determination
 Enthalpy (ΔH), heat capacity (ΔCp) of denaturation
 Characterisation of membranes, lipids, nucleic acids
and micellar systeme
 Equipment: VP-DSC (Malvern)
 Solution heated/cooled from 10-130 oC
Isothermal Titration Calorimetry
 Enzyme kinetics, biological activity or the effect
of molecular structure changes on binding
mechanism
 Complete thermodynamic profile of the molecular
interaction in a single experiment (stoichiometry,
Ka, enthalpy ∆H and entropy ∆S values) or kinetics
parameters Km and kcat
 characterization of biomolecular interactions of
small molecules, proteins, antibodies, nucleic
acids, lipids and others
 Equipment: VP-iTC, iTC200, AutoiTC200
(Malvern Instrum.)
VP-DSC
VP-ITC Auto-iTC200 iTC200
28th Jan 2015 S2004 - Isothermal titration calorimetry
Isothermal Titration Calorimetry
 receptor-ligand interactions
interaction of small molecules
protein-protein interactions
nucleid acid interactions
…..others
 changes in protein ionisation on
binding
 critical micelle concentrations for
detergents
 enzyme kinetics
Applications / Advantages
28th Jan 2015 S2004 - Isothermal titration calorimetry
 Experimental biological relevance
 Label-free
 In-solution
 No molecular weight limitations
 Optical clarity unimportant
 Minimal assay development
 Problematic low affinity interactions
 Sample consumption …
Isothermal Titration Calorimeter
Microcal VP-ITC (Malvern Instruments)
28th Jan 2015 S2004 - Isothermal titration calorimetry
Instrumentation
28th Jan 2015 S2004 - Isothermal titration calorimetry
Reference cell
Thermal jacket
maintains constant
temperature
Ligand in the syringe
Receptor in the cell
Adiabatic jacket
Both cells are from hastelloy alloy
28th Jan 2015 S2004 - Isothermal titration calorimetry
Instrumentation
A constant temperature is controlled by two
main heaters - one for each cell.
Each heater is controlled by a power feedback
sensor.
In case of exothermic reaction - the sample
cell gets warmer than reference cell - less
power supplied to sample cell heater
ITC monitors these heat changes by
measuring the differential power,
applied to the cell heaters
Power feedback sensor which detects
temperature difference between sample and
reference cell and control the temperature
maintenance
Goal T0
Feedback to the
heaters which
compenstate the
temperature
difference
Reference calibration heater
Sample calibration heater
Cell main heater
The Raw ITC Data
28th Jan 2015 S2004 - Isothermal titration calorimetry
28th Jan 2015 S2004 - Isothermal titration calorimetry
In the calorimetric experiment, ligand is titrated to the sample cell
(receptor sample) in a number of small aliquots.
The Raw ITC Data
28th Jan 2015 S2004 - Isothermal titration calorimetry
In the calorimetric experiment, ligand is titrated to the receptor in
the sample cell in a number of small aliquots.
When substances
bind, heat is either
generated or
absorbed.
The Raw ITC Data
Start of titration
large peaks – lots of complex formed on each injection
equal height – virtually every ligand molecule becomes bound to receptor
Raw ITC data is a measure of the power difference supplied to each cell
28th Jan 2015 S2004 - Isothermal titration calorimetry
The Raw ITC Data
The raw signal in the power compensation
calorimeter is the power (cal/sec) applied
to the control heater that is required to
keep the calorimeter cell from changing
temperature as a function of time.
28th Jan 2015 S2004 - Isothermal titration calorimetry
Start of titration
large peaks – lots of complex formed on each injection
equal height – virtually every ligand molecule becomes bound to receptor
The Raw ITC Data
Raw ITC data is a measure of the power difference supplied to each cell
28th Jan 2015 S2004 - Isothermal titration calorimetry
Start of titration
large peaks – lots of complex formed on each injection
equal height – virtually every ligand molecule becomes bound to receptor
The Raw ITC Data
Raw ITC data is a measure of the power difference supplied to each cell
28th Jan 2015 S2004 - Isothermal titration calorimetry
Around equivalence point
heat change decreases as binding sites fill up
Start of titration
large peaks – lots of complex formed on each injection
equal height – virtually every ligand molecule becomes bound to receptor
The Raw ITC Data
Raw ITC data is a measure of the power difference supplied to each cell
28th Jan 2015 S2004 - Isothermal titration calorimetry
End of titration
all binding sites occupied – no further binding
only “dilution peaks” after addition of more ligand
Start of titration
large peaks – lots of complex formed on each injection
equal height – virtually every ligand molecule becomes bound to receptor
Around equivalence point
heat change decreases as binding sites fill up
The Raw ITC Data
Raw ITC data is a measure of the power difference supplied to each cell
The pattern of the heat effects/mol of
titrant as a function of the molar ratio
[ligand]/[macromolecule] can then be
analysed to give the thermodynamic
parameters of the interaction.
Evaluation of ITC Data
These heat flow peaks are integrated with
respect to time, giving the total heat
released/absorbed after each injection
point.
28th Jan 2015 S2004 - Isothermal titration calorimetry
H = Q / concentration of titrant
The pattern of the heat effects/mol of
titrant as a function of the molar ratio
[ligand]/[macromolecule] can then be
analysed to give the thermodynamic
parameters of the interaction.
Evaluation of ITC Data
These heat flow peaks are integrated with
respect to time, giving the total heat
released/absorbed after each injection
point.
28th Jan 2015 S2004 - Isothermal titration calorimetry
Stoichiometry
Affinity
Enthalpy
In one ITC experiment:
 Enthalpy H
 Equilibrium binding constant Ka
 Stoichiometry
P + L PLKa
Kd
∆G = -RT ln Ka
∆G = ∆H - T∆S
…. calculate:
Kd = 1 / Ka
28th Jan 2015 S2004 - Isothermal titration calorimetry
Evaluation of ITC Data
ligand / macromolecule
28th Jan 2015 S2004 - Isothermal titration calorimetry
Receptor - ligand interactions
 
  XM
MX
aK
High affinity = large Ka, small Kd
fast association, slow dissociation
  
 MX
XM
dK
[L / mol]
[mol / L]
i.e. Kd is a concentration
Receptor - ligand interactions
M X MX
+
28th Jan 2015 S2004 - Isothermal titration calorimetry
Ka
Kd
Kd = 1 / Ka
M X MX
Receptor - ligand interactions
+
∆G = -RT ln Ka
∆G = ∆H - T∆S
Free energy Enthalpy Entropy
28th Jan 2015 S2004 - Isothermal titration calorimetry
Ka
Kd
∆G = ∆H - T∆S
G ≤ 0 for spontaneous process
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G and spontaneous processes
High affinity = high Ka, low Kd, high -G
M X MX
Receptor - ligand interactions
+ - water molecules
- ionts
- protons
28th Jan 2015 S2004 - Isothermal titration calorimetry
Enthalpy : System has a tendency to reach the
minimum energetic state.
H has tendency to be negative.
….bonds are formated
Entropy : At the molecular level, Brown´s motion
rises the entropy. Entropy rises with temperature.
TS has tendency to be positive.
Formation of bonds means that entropy is
decreasing
∆G = ∆H - T∆S
Enthalpy
Changes in heat
Structure of the complex
 Hydrogen bonding
 Van der Waals
Structure of the solvent
 i.e. water
Enthalpy change - energy content of the
bonds broken and created. The dominant
contribution is from hydrogen bonds.
Negative value indicates enthalpy change
favoring the binding.
Negative is favourable
Entropy
Changes in disorder
Independent rotational and translational
degrees of freedom
 A complex is less disordered than two
molecules
Internal conformational dynamics
 Flexible molecules lose entropy in the
process binding
Dynamics of the solvent
 i.e. water
Bonds formation means higher order of the
system therefore entropy decreases
Positive favourable
∆G = ∆H - T∆S
28th Jan 2015 S2004 - Isothermal titration calorimetry
Unfavorable enthalpy positive for entropically
driven reactions:
Hydrophobic interactions
Solvation entropy due to release of water
 Hydrophobic interaction: mostly characterized by less positive enthalpy, but positive
entropy changes, due to the solvent reorganization around the nonpolar groups.
 Electrostatic interactions: entropically driven with less changes in enthalpy.
 Hydrogen bonds: enthalpically driven (estimated energy of one hydrogen bond is 5kcal/mol)
 Conformational changes: less changes in the enthalpy and entropy contributions.
 Water molecules: Structured water molecules / bulk water molecules
Water molecules released from the protein binding site – this process is characterized by
increasing of entropy, and the process is also enthalpically unfavourable.
Characterization of interaction
Hydrogen bonds
Hydrophobic region
Water molecules
Protein
Rotation
and
flexibility
of ligand
A
B
A
B
28th Jan 2015 S2004 - Isothermal titration calorimetry
Receptor - ligand interactions
Back to the ITC Data
0 1 2 3 4 5 6
-0,20
-0,15
-0,10
-0,05
0,00
-0,60
-0,40
-0,20
0,00
0,20
0 10 20 30 40 50 60 70
Time (min)
µcal/sec
Molar Ratio
KCal/MoleofInjectant
0,0 0,5 1,0 1,5
-8,00
-6,00
-4,00
-2,00
0,00
-1,00
-0,80
-0,60
-0,40
-0,20
0,00
0,20
-10 0 10 20 30 40 50 60 70 80 90
Time (min)
µcal/sec
Molar Ratio
KCal/MoleofInjectant
0 1 2 3 4 5
0,00
0,50
1,00
1,50
2,00-0,50
0,00
0,50
1,00
1,50
2,00
2,50
3,00
0 10 20 30 40 50 60 70
Time (min)
µcal/sec
Molar Ratio
KCal/MoleofInjectant
Ka 103 - 109 M-1 (Kd - 1 mM – 10 nM)
High affinity Low affinity
Ka 105 M-1
Ka 103 M-1
28th Jan 2015 S2004 - Isothermal titration calorimetry
ITC Data
0 1 2 3 4 5 6
-0,20
-0,15
-0,10
-0,05
0,00
-0,60
-0,40
-0,20
0,00
0,20
0 10 20 30 40 50 60 70
Time (min)
µcal/sec
Molar Ratio
KCal/MoleofInjectant
0,0 0,5 1,0 1,5
-8,00
-6,00
-4,00
-2,00
0,00
-1,00
-0,80
-0,60
-0,40
-0,20
0,00
0,20
-10 0 10 20 30 40 50 60 70 80 90
Time (min)
µcal/sec
Molar Ratio
KCal/MoleofInjectant
0 1 2 3 4 5
0,00
0,50
1,00
1,50
2,00-0,50
0,00
0,50
1,00
1,50
2,00
2,50
3,00
0 10 20 30 40 50 60 70
Time (min)
µcal/sec
Molar Ratio
KCal/MoleofInjectant
Ka 103 - 109 M-1 (Kd - 1 mM – 10 nM)
High affinity Low affinity
Ka 105 M-1
Ka 103 M-1
28th Jan 2015 S2004 - Isothermal titration calorimetry
ITC Data
Generally….
c > 10
sigmoidal curve that becomes
steeper as c increases
c < 10
curve becomes flatter
The curve shape depends on the “c-value”
M = c / n x Ka
c = n x M / Kd
28th Jan 2015 S2004 - Isothermal titration calorimetry
For high affinity ligands
c > 500
[M]total >> Kd
slope is too steep to determine Kd
only H and n can be measured
For very high affinity ligands (low
Kd) must use low macromolecule
concentration
But low [M] gives very small
signals…
Therefore Kd limit = 10 nM
28th Jan 2015 S2004 - Isothermal titration calorimetry
The curve shape depends on the “c-value”
M = c / n x Ka
c = n x M / Kd
Low affinity ligands
c < 1
[M]total << Kd
curve becomes very flat
For very low affinity ligands (high Kd)
must use high macromolecule
concentration
But proteins often soluble to only 1
mM…
Therefore Kd limit = 1 mM
(Reverse titration ???)
Or must add many equivalents of
ligand…
Kd limit = 100 mM?
STOICHIOMETRY !!!
28th Jan 2015 S2004 - Isothermal titration calorimetry
The curve shape depends on the “c-value”
M = c / n x Ka
c = n x M / Kd
Very high and very low affinity systems can be studied using
DISPLACEMENT TITRATIONS
 High affinity ligand added to a solution of the low affinity complex
 High affinity ligand displaces the low affinity ligand
 Change in the apparent affinity and apparent enthalpy
 If parameters for one ligand are known, possible to calculate for the other ligand
+ +
Low affinity complex High affinity complex
28th Jan 2015 S2004 - Isothermal titration calorimetry
28th Jan 2015 S2004 - Isothermal titration calorimetry
ITC experimental set-up
 Set of titrations (continue
injections)
– Direct titration
– Reverse Titration
 Competitive binding
 Single injection
 Set of titrations (continue
injections)
– Direct titration
– Reverse Titration
 Competitive binding
 Single injection
28th Jan 2015 S2004 - Isothermal titration calorimetry
ITC experimental set-up
0,0 0,5 1,0 1,5
-8,00
-6,00
-4,00
-2,00
0,00
-1,00
-0,80
-0,60
-0,40
-0,20
0,00
0,20
-10 0 10 20 30 40 50 60 70 80 90
Time (min)
µcal/sec
Molar Ratio
KCal/MoleofInjectant
0 1 2 3 4 5 6
-0,20
-0,15
-0,10
-0,05
0,00
-0,60
-0,40
-0,20
0,00
0,20
0 10 20 30 40 50 60 70
Time (min)
µcal/sec
Molar Ratio
KCal/MoleofInjectant
+ +
Low affinity complex High affinity complex
Low affinity complex High affinity complex
28th Jan 2015 S2004 - Isothermal titration calorimetry
ITC experimental set-up
 Set of titrations (continue
injections)
– Direct titration
– Reverse Titration
 Competitive binding
 Single injection
Sample preparation
 The concentration of both samples must be determined preciselly
 Samples must be in the exactly same buffer (heat of dillution)
 Dissolve your samples in lyophilisate form in the working buffer
– Samples must be dialyzed exhaustively against working buffer
28th Jan 2015 S2004 - Isothermal titration calorimetry
 For the first experiment (Kd is not known) 10 times higher concentration of
ligand is recommended
 Minimal concentration of macromolecule presenting in the calorimetric cell
is 10 M
 pH of the samples must be checked carefully
 Blank measurement
 Filtration and degassing of the samples
28th Jan 2015 S2004 - Isothermal titration calorimetry
Troubleshooting
28th Jan 2015 S2004 - Isothermal titration calorimetry
Buffer Mismatch
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Buffer Mismatch
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Blank experiment subtraction
28th Jan 2015 S2004 - Isothermal titration calorimetry
Air bubble / cleanless
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Not enough time between injections
28th Jan 2015 S2004 - Isothermal titration calorimetry
28th Jan 2015 S2004 - Isothermal titration calorimetry