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)-
calorimeter 열량계

5. Thermometric Titration
A number of instrumental titration techniques
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
온도 변화에 따른 열에너지 변화를 측정
고분자 물질 연구에 많이 이용
시료물질과 기준물질을 동시에 가열/냉각시켜 시료의 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)
결정화 시간, 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 : First Injection

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 요인에 대한 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 비 가역과정을 나타내고 등호는 가역과정을 나타낸다.

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

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)
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 the Van't Hoff Equation

42. Van't Hoff equation(1/2) 평형 상수의 자연로그와 온도의 역수 값에 대한 그래프는 직선을 그린다.
이 직선의 기울기는 엔탈피의 변화량을 기체상수로 나누어준 값의 음의 값이다.
절편값은 엔트로피의 변화량을 기체상수로 나누어준 값이다.
이 식을 미분형태로 표현한 것이 Van't Hoff Equation이다.

43. Van't Hoff equation(2/2)
온도 변화에 따른 평형상수(K)의 변화 비를 엔탈피 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 Retrieval system Online
Bibliographic database of life science and biomedical information
Medicine, nursing, pharmacy, dentistry, health care, biology, biochemistry and molecular evolution
Searchable via PubMed

48. Data Analysis

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

50. Summary

51. Advantages / Disadvantages
Immobilization or labeling이 필요 없다.
다양한 적용 범위
Kd, ΔH 측정
다른 온도와 pH에서 가능
Disadvantages
Enormous amounts of binding partner
Only medium affinity
많은 membrane proteins에 제약
비싼 가격

53. Summary Thermodynamic parameters Protein-ligand 영역으로의 확장
Characterization and understanding of chemical
reaction
Protein-ligand 영역으로의 확장
Drug-discovery등의 다양한 영역에 실용적
이전 van't Hoff technique에서 발전
Modern, automated, high-sensitivity calorimetry equipment
Proteinomics 관심 대상
Biomolecules의 folding이나 ligands의 결합