ITC (Isothermal Titration Calorimetry )

1. ITC(Isothermal Titration Calorimetry) 황정현

2. Contents • Introduction • ITC technology • Principle • Application • Data analysis • Summary

3. Introduction

4. Introduction • Isothermal Titration Calorimetry • Iso- : 같은, 동(同)-, 등(等)- • Thermal : ‘열’의 • Titration : 적정 • Calorimetry : 열량 측정 • calorimeter 열량계

5. Thermometric Titration • Anumberofinstrumentaltitrationtechniques • Accurate and precise without a subjective interpretation • Reported early in the 20th century • (Bell and Cowell, 1913)

6. ITC Technology

7. ITC technology • Calometer를 이용한 the heat of a reaction 측정법 • 현재, engineering과 computer 기술이 적용. 자동화된 software를 사용 • DSC (differential scanning calorimeter) • ITC (isothermal titration calorimeter)

8. Differential Scanning Calorimeter (1/4) • 시차주사 열량법 • 온도변화에따른열에너지변화를측정 • 고분자물질연구에많이이용 • 시료물질과 기준물질을 동시에 가열/냉각시켜시료의 열 출입을 측정 • 기준물질:가열로의 온도조절에 따라 • 시료물질:주어지는 온도에 의해 • Heat flux plate에 의해 열량 값을 얻음

9. Differential Scanning Calorimeter (2/4)

10. Differential Scanning Calorimeter (3/4)

11. Differential Scanning Calorimeter (4/4)

12. DSC thermogram (1/2) • Glass transition temperature(Tg) • Melting temperature(Tm) • Crystallization temperature(Tc) • 결정화시간, 순도, 산화, 분해

13. DSC thermogram (2/2)

14. Isothermal Titration Calorimeter (1/6) • Biomolecular interactions에 관한 연구 • protein-ligand • protein-DNA • Antibody-antigen • Hormone-receptor

15. Isothermal Titration Calorimeter (2/6) • 모든 binding parameter를 측정 • Binding이 나타날 때 • Heat is taken up • Absorbed • endothermic • Heat is evolved • Released • exothermic

16. Isothermal Titration Calorimeter (3/6)

17. Isothermal Titration Calorimeter (4/6) • Reference and sample cell are identical. • Aliquots of the second binding partner are added with a stirring syringe • The sample cell is mixed with stirring paddles at the syringe tip.

18. Isothermal Titration Calorimeter (5/6) • The reference cell is electrical heated preset steady temperature. • The temperature difference between reference and sample cell is measured.

19. Isothermal Titration Calorimeter (6/6)

20. ITC – Before Titration

21. Titration Begins : FirstInjection

22. Return to Baseline

23. Second Injection

24. Second Return to Baseline

25. Injections Continue

26. Injections Continue

27. End of Titration

28. ITC – Fitting the Data (1/3)

29. ITC – Fitting the Data (2/3) • The peaks from the upper panel raw data are integrated plotted with respect to the concentrations of the interacting components as molar heats(y-axis) and molar ratio(x-axis).

30. ITC – Fitting the Data (3/3) • Fitting of this curve gives the parameters derived in the text.

31. Principle

32. Principle • Biological macromolecules의 interaction • Molecular recognition의 complexity and diversity • Immune response, signal transduction cascades, gene expression등 중요 요인에대한 관심과 적용

33. Basic Thermodynamics (1/9) • 연관변수를 측정하여 대상의 정체를 확인 • n : Stoichiometry of the interaction • Ka: Association constant • Kd: Dissociation constant • ΔGb :Free energy • ΔHb:Enthalpy • ΔSb:Entropy • ΔCp:Heat capacity of binding

34. Basic Thermodynamics (2/9) • At Protein-Ligand Interactions • The First Law of Thermodynamics • 열역학 제 1법칙 • ΔE=Q+W • ΔE represents the change in the energy • Q the heat absorbed by the system • W the work done on the system

35. Basic Thermodynamics (3/9) • At Protein-Ligand Interactions • The Second Law of Thermodynamics • 열역학 제 2법칙 • 고립계에서 총 entropy(무질서도)의 변화는 항상 증가하거나 일정하며 절대로 감소하지 않는다. 에너지는 방향이 있다는 것이다. • ΔS≥0 • 부등호는 비가역과정을 나타내고등호는 가역과정을 나타낸다.

36. Basic Thermodynamics (4/9) • At Protein-Ligand Interactions • The Second Law of Thermodynamics or • By defining change in “Entropy” as or

37. Basic Thermodynamics (5/9) • At Protein-Ligand Interactions • 대부분의 protein-ligand interactions • At constant temperature & Pressure • Only work is –PΔV • We can change this term to ΔH, then

38. Basic Thermodynamics (6/9) • With the definition of (Gibbs) 'Free Energy' as • ΔG＜0 : spontaneous change • ΔG = 0 : Equilibrium

39. Basic Thermodynamics (7/9) • ΔH의 효용성 • Direct measurement of heat of reaction • No ΔPV-work is the same as ΔH

40. Basic Thermodynamics (8/9) • Indirect measure • Utilizes a simplified relationship • The Van't Hoff Equation • Gibbs Free Energy Equation • At steady state, at which ΔG=0, then

41. Basic Thermodynamics (9/9) • Gibbs Free Energy Equation • This is an integrated form of theVan't Hoff Equation

42. Van't Hoff equation(1/2) • 평형 상수의 자연로그와 온도의 역수 값에 대한그래프는 직선을 그린다. • 이 직선의 기울기는 엔탈피의변화량을기체상수로 나누어준 값의 음의 값이다. • 절편값은 엔트로피의 변화량을 기체상수로나누어준 값이다. • 이 식을 미분형태로 표현한 것이Van't Hoff Equation이다.

43. Van't Hoff equation(2/2) • 온도 변화에 따른 평형상수(K)의 변화 비를엔탈피 변화를 이용하여 표현

44. Application

45. Application • 실험 data는 protein-ligand 연구정보를 참고하여 분석 • MEDLINE search • ITC equipment suppliers

46. MEDLINE • Medical Literature Analysis and Retrievalsystem Online • Bibliographic database of life science andbiomedical information • Medicine, nursing, pharmacy, dentistry,health care, biology, biochemistry andmolecular evolution • Searchable via PubMed

48. Data Analysis

49. 생물리학 연계성 • Thermodynamic parameters를 측정 • 생체 물질의 interaction • Drug나 Enzyme에 관련해서 직접적으로 • 축적된 3-D protein structures의 이해 • 여러 가지 결합 상황을 예측, design 가능 • Weak forces로 이루어지는 protein-ligandinteraction을 분석, 추정

50. Summary