ITC
(Isothermal Titration Calorimetry)
황정현
Contents
Introduction
ITC technology
Principle
Application
Data analysis
Summary
Introduction
Introduction
Isothermal Titration Calorimetry
Iso- : 같은, 동(同)-, 등(等)Thermal : ‘열’의
Titration : 적정
Calorimetry : 열량 측정
calorimeter 열량계
Thermometric Titration
A number of instrumental titration techniques
Accurate and precise without a subjective int
erpretation
Reported early in the 20th century
(Bell and Cowell, 1913)
ITC Technology
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)
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
Differential Scanning Calorimeter
(2/4)
Differential Scanning Calorimeter
(3/4)
Differential Scanning Calorimeter
(4/4)
DSC thermogram (1/2)
Glass transition temperature(Tg)
Melting temperature(Tm)
Crystallization temperature(Tc)
결정화 시간, 순도, 산화, 분해
DSC thermogram (2/2)
Isothermal Titration Calorimeter (1/6)
Biomolecular
interactions에 관한
연구
protein-ligand
protein-DNA
Antibody-antigen
Hormone-receptor
Isothermal Titration Calorimeter (2/6)
모든 binding
parameter를 측정
Binding이 나타날 때
Heat is taken up
- Absorbed
- endothermic
Heat is evolved
- Released
- exothermic
Isothermal Titration Calorimeter (3/6)
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.
Isothermal Titration Calorimeter (5/6)
The reference cell is
electrical heated
preset steady
temperature.
The temperature
difference between
reference and
sample cell is
measured.
Isothermal Titration Calorimeter (6/6)
ITC – Before Titration
Titration Begins :
Injection
First
Return to Baseline
Second Injection
Second Return to Baseline
Injections Continue
Injections Continue
End of Titration
ITC – Fitting the Data (1/3)
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(xaxis).
ITC – Fitting the Data (3/3)
Fitting of this curve
gives the
parameters derived
in the text.
Principle
Principle
Biological macromolecules의 interaction
Molecular recognition의 complexity and
diversity
Immune response, signal transduction
cascades, gene expression등 중요 요인에
대한 관심과 적용
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
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
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.
Basic Thermodynamics
(4/9)
At Protein-Ligand Interactions
The Second Law of Thermodynamics

Q 
 0
T 
or
 Q reversible 
 d  T   0
By defining change in “Entropy” as
 S
system

 S
surroundin g
 0 or
 dS
0
S 
Q
T
Basic Thermodynamics
(5/9)
At Protein-Ligand Interactions
대부분의 protein-ligand interactions
At constant temperature & Pressure
Only work is –PΔV
 S system 
 E
 PV
system
0
T
E  PV
We can change this term to ΔH, then
S 
 H 
T
 0
TS  H  0
Basic Thermodynamics
(6/9)
With the definition of (Gibbs) 'Free Energy'
as
G  H  TS
ΔG＜ 0 : spontaneous change
ΔG = 0 : Equilibrium
 G   RT ln K b   H  T  S
Basic Thermodynamics
(7/9)
ΔH의 효용성
Direct measurement of heat of reaction
No ΔPV-work is the same as ΔH
 E   H   PV
 E  H
Basic Thermodynamics
(8/9)
Indirect measure
Utilizes a simplified relationship
The Van't Hoff Equation
P  L  PL
K
Gibbs Free Energy Equation
 PL  
 G   G  RT ln 

 P L  
0
At steady state, at which ΔG=0, then
 PL  
 G   RT ln 

 P L  
0
eq

PL 
P L 
Basic Thermodynamics
(9/9)
Gibbs Free Energy Equation
 G   RT ln K eq
0
 H
ln  K d   
 R
0
 1 
   RT ln    RT ln  K d
 Kd 
 1   S 0
  
 T
R
 
This is an integrated form of the
Van't Hoff Equation
d ln K eq 
dT

H
0
RT
2

Van't Hoff equation(1/2)
 H
ln  K d   
 R
0
 1   S 0
  
 T
R
 
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.
Van't Hoff equation(2/2)
온도 변화에 따른 평형상수(K)의 변화 비를
엔탈피 변화를 이용하여 표현
d ln K eq 
dT

H
0
RT
2
Application
Application
실험 data는 protein-ligand 연구정보를
참고하여 분석
MEDLINE search
ITC equipment suppliers
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
Data Analysis
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
Summary
Advantages / Disadvantages
Advantages
Disadvantages
Immobilization or
Enormous amounts
labeling이 필요 없다.
of binding partner
다양한 적용 범위
Only medium affinity
Kd, ΔH 측정
많은 membrane
다른 온도와 pH에서
proteins에 제약
가능
비싼 가격
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