ITC
(Isothermal Titration Calorimetry)
Contents
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Introduction
ITC technology
Principle
Application
Data analysis
Summary
Introduction
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Isothermal Titration Calorimetry
Iso- : 같은, 동(同)-, 등(等)Thermal : ‘열’의
Titration : 적정
Calorimetry : 열량 측정
– calorimeter 열량계
Introduction
Thermodynamics!
• The island of incomprehensibility!
Covered by a jungle of strange
terminology wherein the biologist
hacked through the underbrush of lush
formulas for the prelims, just to paddle
across the ocean of amnesia as quickly
as possible (never to return).
Introduction
Term.
• Stoichiometry of the interaction (n)
• Association constant (Ka) /
Dissociation constant (Kd)
• Free energy (ΔGb)
• Enthalpy (ΔHb)
• Entropy (ΔSb)
• Heat capacity of binding (ΔCp)
ITC technology
• Calometer를 이용한 the heat of a
reaction을 측정법
• 현재, engineering과 computer 기술이 적
용. 자동화된 software를 사용
• DSC (differential scanning calorimeter)
• ITC (isothermal titration calorimeter)
ITC technology
DSC
(Differential Scanning Calorimeter)
• Ideal for stability and folding studies
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측정
Tm (Transition midpoint)
Enthalpy (ΔH)
Heat capacity (ΔCp)
ITC technology
ITC
(Isothermal Titration Calorimeter)
• Biomolecular interactions에 관한 연구
– (protein-ligand ; protein-DNA, antibodyantigen, hormone-receptor)
• 모든 binding parameter를 측정
• Binding이 나타날 때
– Heat is taken up (absorbed, endothermic)
– Heat is evolved (released, exothermic)
Principle
• Biological macromolecules의 interaction
• Molecular recognition의 complexity and
diversity
• Immune response, signal transduction
cascades, gene expression등 중요 요인
에 대한 관심과 적용
Principle
Thermodynamics 연관 변수 측정하여 대상
의 정체를 밝히고자 한다.
• Stoichiometry of the interaction (n)
• Association constant (Ka)
/
Dissociation constant (Kd)
• Free energy (ΔGb)
• Enthalpy (ΔHb)
• Entropy (ΔSb)
• Heat capacity of binding (ΔCp)
Principle
Basic Thermodynamics of
Protein-Ligand Interactions
• The First Law of Thermodynamics
• ΔE=Q+W
– ΔE represents the change in the energy
– Q the heat absorbed by the system
– W the work done on the system
Principle
• The Second Law of Thermodynamics
Q
  T   0 or
 Qreversible 
 d  T   0
Q
by defining change in "entropy" as S 
T
S
or
system
  Ssurroundin g  0
 dS  0
Principle
• 대부분 protein-ligand interactions
– Constant temperature and pressure에서
– only work is –PΔV
S system 
E  PV system
T
0
E  PV is the change in "enthalpy"
S 
H   0
T
TS  H  0
Principle
• With the definition of
(J.Willard Gibbs) "free energy" as
G  H  TS
– ΔG<0 spontaneous change
– ΔG=0 equilibrium
G  RT ln Kb  H  TS
Principle
ΔH의 효용성
• Direct measurement of the heat of
reaction
– No △PV-work is the same as △H
E  H  PV
 E  H
Principle
• Indirect measure
– utilizes a simplified relationship
– (the van't Hoff equation)
 PL 
G  G  RT ln

 PL 
0
at steady state, at which ΔG=0
 PL 
G   RT ln

 PL 
0
P  L  PL

P L
K eq 
P L
Principle
 1
G   RT lnK eq    RT ln
 Kd
 H 0  1  S 0
  
lnK d   
R
 R  T 
0

  RT lnK d 

• This is an integrated form of
the van't Hoff equation
d ln K eq 
dT
H 0

2
RT
Application
Application
• 실험 data는 protein-ligand 연구정보를
참고하여 분석
• MEDLINE search
• ITC equipment suppliers
Application
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-ligand
interaction을 분석, 추정
Application
Advantages / Disadvantage
Immobilization or
labeling 필요 없다.
다양한 적용 범위
Kd, ΔH 측정
다른 온도와 pH에서
가능
◊ Enormous amounts
of binding partner
◊ Only medium affinity
◊ 많은 membrane
proteins에 제약
◊ 비싼 가격
Summary
Summary
• Thermodynamic parameters
– 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