Characterising complex binding interactions by ...
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Characterising complex binding interactions by microcalorimetry
Transcript of Characterising complex binding interactions by ...
Characterising complex binding interactions by microcalorimetry
Isothermal Titration Calorimetry &
Differential Scanning Calorimetry
Articles written with isothermal titration calorimetry
content since 1990 sourced from the Web of Science ™.
Falconer, R.J. (2016) Applications of isothermal titration calorimetry - the research and technical developments
from 2011-15. Journal of Molecular Recognition, 29: 504–515.
Differential Scanning Calorimetry
20 40 60 80 100-0.005
0.000
0.005
0.010
0.015
First scan
Subsequent scans
Temperature (Celsius)
Heat
cap
acit
y (
kJ/K
)
Thermal scan of grape invertase
0 20 40 60 80-0.0010
-0.0005
0.0000
0.0005
0.0010First scan
Subsequent scans
Temperature (Celsius)
Heat
cap
acit
y (
kJ/K
)
Thermal scan of grape thaumatin-like protein using a DSC
Differential Scanning Calorimetry
Sanchez-Ruiz, et al. Biochemistry, 1988, 27, 1648-1652.
k = v Cp / (Qt - Q)
0.00300 0.00305 0.00310 0.00315 0.00320-6
-5
-4
-3
-2
-1
0
1
2
1/T
lnk
Arrhenius Equation
k = A exp(-E/RT)
Estimate the activation energy of unfolding
0 10 20 30 40-0.024
-0.023
-0.022
-0.021
-0.020
Temperature (ºC)
He
at
cap
acit
y (k
J/°
C)
0 10 20 30 40-0.024
-0.023
-0.022
-0.021
-0.020
Temperature (ºC)
He
at
cap
acit
y (k
J/°° °°
C)
Differential scanning calorimetry of cryoglobulins (a) the downward thermal scan showing cold-induced
aggregation, (b) upward thermal scan showing heat-induced disassociation, the cryoglobulin Pot IgM
(black) and Yvo IgM (green).
Thermal scan of IGM cold-induced precipitation using a DSC
Differential Scanning Calorimetry
Meliga, S.C.; Farrugia, W.; Ramsland, P.A.; Falconer, R.J. (2013) Cold-induced precipitation of a monoclonal IgM; a negative
activation enthalpy reaction. Journal of Physical Chemistry B, 117: 490-494.
Thermal scan of grape lipid-transfer protein using a DSC
Differential scanning calorimetry of grape lipid-transfer protein purified from Vitis vinifera
Differential Scanning Calorimetry
Differential Scanning Calorimetry
Pressure perturbation calorimetry of sodium dihydrogen phosphate and isopropanol solutions.
Pressure perturbation calorimetry using a DSC
Bye, J.W.; Freeman, C.L.; Howard, J.D.; Herz, G.; McGregor, J.; Falconer, R.J. (2017) Pressure perturbation calorimetry analysis of
the mesoscopic structuring of 2-propanol/water mixtures. Journal of Solution Chemistry, 46: 175-189.
Isothermal Titration Calorimetry
Stoichiometry
Association constant Ka
Thermodynamic constants
ΔH, TΔS & ΔG
ΔG = -RTlnKa
ΔG = ΔH - TΔS
McRae, J.M.; Falconer, R.J.; Kennedy, J.A. (2010) Thermodynamics of
grape and wine tannin interaction with polyproline: implications for
red wine astringency. Journal of Agricultural and Food Chemistry, 58:
12510–12518.
HO O
OH
OH
R1
OH
R2
R3
O
O
OH
OH
OH
1 R1=OH, R2,R3=H2 R1,R3=H, R2=OH3 R1=Galloyl, R2=OH, R3=H4 R1=H, R2,R3=OH
Galloyl=
HO+O
OH
OH
OH
OH
OGlucose
5
2
4
8
6
B
A
N
NO
O
n
O
OH
O
OH
O
OH
HO
HO
OH
OH
OH
OH
OH
OH
OH
OH
OH
HO
O
OH
O
OH
O
HO
HO
O
OH
OH
HO
HO
OH
OH
OH
OH
O
OOH
OH
O
OH
HO
O
OH
HO
OH
OH
HO
O
OH
OH
OH
OHHO
O
OH
OH
OH
OHHO
a. b.
Polyphenolics – Polyproline Binding
Wine polyphenolics – Polyproline Binding
McRae, J.M.; Falconer, R.J.; Kennedy, J.A. (2010) Thermodynamics of grape and wine tannin interaction with
polyproline: implications for red wine astringency. Journal of Agricultural and Food Chemistry, 58: 12510–12518.
Dual interactions
Hydrophobic interaction and H-bonding
10 ºC 45 ºC
Polyphenolics – BSA Binding
Isothermal titration calorimetry of the interaction of the proanthocyanidin trimer, pentamer, hexamer
with BSA showing the thermogram and binding isotherm. All at pH 4.0 in a 10 mM ammonium acetate
buffer at 10 oC.
Kilmister, R.L.; Faulkner, P.; Downey, M.O.; Darby, S.J.; Falconer, R.J. (2016) The complexity of condensed tannin binding to
bovine serum albumin - An isothermal titration calorimetry study. Food Chemistry, 190: 173-178.
Polyphenolics – BSA Binding
Isothermal titration calorimetry of the interaction of the proanthocyanidin tetramer with BSA at different
temperatures a) 10 oC and b) 25 oC and c) 10 oC with 100 mM NaCl showing the thermogram and binding
isotherm. All at pH 4.0 in a 10 mM ammonium acetate buffer.
Kilmister, R.L.; Faulkner, P.; Downey, M.O.; Darby, S.J.; Falconer, R.J. (2016) The complexity of condensed tannin binding to
bovine serum albumin - An isothermal titration calorimetry study. Food Chemistry, 190: 173-178.
Electrostatic interaction – Phytate with lysozyme
Electrostatic interaction – Phytate with lysozyme
Isothermal titration calorimetry (ITC) titration of phytate (6mM) into lysozyme (0.4mM) in 10mM
ammonium acetate buffer pH 4 at 25 °C showing the thermogram (top) and binding isotherm (bottom).
Electrostatic interaction – Phytate with lysozyme
Isothermal titration calorimetry (ITC) titration of phytate (6mM) into lysozyme (0.4mM) in 10mM
ammonium acetate buffer pH 4 at 40 °C showing the thermogram (top) and binding isotherm (bottom).
Electrostatic interaction – Phytate with lysozyme
Darby, S.J.; Platts, L.; Daniel, M.S.; Cowieson, A.J.; Falconer, R.J. (2017) An isothermal titration calorimetry study of phytate
binding to lysozyme: A multisite electrostatic binding reaction. Journal of Thermal Analysis and Calorimetry, 127: 1201–
1208.
Isothermal Titration Calorimetry
Lambert, F.L. (2002) Entropy is simple, qualitatively. Journal of Chemical Education, 79: 1241-1246.
Leff, H.S. (1996) Thermodynamic entropy: The spreading and sharing of energy. American Journal of
Physics, 64: 1261-1271.
Falconer, R.J. (2016) Applications of isothermal titration calorimetry - the research and technical
developments from 2011-15. Journal of Molecular Recognition, 29: 504–515.
What have we learnt?
Binding interactions involve water displacement
Water displacement is usually endothermic while bond formation is exothermic
ΔHobserved = ΔHbonding + ΔHwater
ΔG = ΔH - TΔS
How do you interpret the entropy values?
For an modern explanation of entropy read Franks Lambert and Harvey Leff’s papers
Thank you
