ITC (Isothermal Titration Calorimetry)

1. ITC(Isothermal Titration Calorimetry)

2. ITC • 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)

3. Biological Thermodynamics • 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

4. Internal Energy (U) • 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

5. Enthalpy (H) 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

6. Enthalpy change (ΔH) • Exothermic reaction • ΔH is negative • Emits heat • Endothermic reaction • ΔH is positive • Absorbs heat

7. Isothermal Titration Calorimetry (ITC)

8. Isothermal Titration Calorimetry (ITC)

9. Isothermal Titration Calorimetry (ITC) • 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

10. Isothermal Titration Calorimetry (ITC)

11. Isothermal Titration Calorimetry (ITC) • Exothermic reaction: • Emit heat • Negative peak on ITV • Endothermic reaction: • Absorb heat • Positive peak on ITC

12. ITC – fitting the data

13. ITC data – binding isotherms for various c values

14. 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).

15. As promised… Thermodynamics

16. First Law of thermodynamics • 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.

17. Enthalpy (H) 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

18. Second Law of thermodynamics • The total entropy of a system and its surroundings always increases for a spontaneous process

19. The Gibbs free energy (ΔG) 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

20. The Gibbs free energy (ΔG) • 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!

21. Biological Thermodynamics • Reactant  Product • Association constant: • ΔG = –RT lnKeq • Keq = 10–ΔG/1.36

22. Biological Thermodynamics at steady state, at which ΔG=0

23. ITC Advantages Disadvantage Enormous amounts of binding partner Only medium affinity Limitation for membrane proteins High price • Immobilization or labeling 필요 없다. • Kd, ΔH can be measured • Can be applied to different reaction temperature and pH