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ITC(Isothermal Titration Calorimetry)
황정현
Contents
Introduction
ITC technology
Principle
Application
Data analysis
Summary
Introduction
IntroductionIsothermal Titration Calorimetry
Iso- : 같은 , 동 ( 同 )-, 등 ( 等 )-
Thermal : ‘ 열’의
Titration : 적정
Calorimetry : 열량 측정
calorimeter 열량계
Thermometric TitrationA number of instrumental titration tech-niques
Accurate and precise without a subjective
interpretation
Reported early in the 20th century
(Bell and Cowell, 1913)
ITC Technology
ITC technologyCalometer 를 이용한 the heat of a reaction
측정법
현재 , engineering 과 computer 기술이 적용 .
자동화된 software 를 사용
DSC (differential scanning calorimeter)
ITC (isothermal titration calorimeter)
Differential Scanning Calorimeter
(1/4)시차주사 열량법
온도 변화에 따른 열에너지 변화를 측정고분자 물질 연구에 많이 이용
시료물질과 기준물질을 동시에 가열 / 냉각시켜시료의 열 출입을 측정
기준물질 : 가열로의 온도조절에 따라
시료물질 : 주어지는 온도에 의해
Heat flux plate 에 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
interactions 에 관한
연구
protein-ligand
protein-DNA
Antibody-antigen
Hormone-receptor
Isothermal Titration Calorimeter (2/6)모든 binding
parameter 를 측정
Binding 이 나타날 때Heat is taken up
- Absorbed
- endothermic
Heat is evolved- Released
- exothermic
Isothermal Titration Calorimeter (3/6)
Isothermal Titration Calorimeter (4/6)Reference and sam-ple cell are identical.Aliquots of the sec-ond binding partner are added with a stirring syringeThe sample cell is mixed with stirring paddles at the sy-ringe 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 : First Injection
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 concentra-tions of the inter-acting components as molar heats(y-axis) and molar ra-tio(x-axis).
ITC – Fitting the Data (3/3)Fitting of this curve
gives the
parameters derived
in the text.
Principle
PrincipleBiological macromolecules 의 interaction
Molecular recognition 의 complexity and
diversity
Immune response, signal transduction cas-
cades, gene expression 등 중요 요인에
대한 관심과 적용
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 (1/9)
At Protein-Ligand Interactions
The First Law of Thermodynamics
열역학 제 1 법칙
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 (2/9)
At Protein-Ligand Interactions
The Second Law of Thermodynamics열역학 제 2 법칙
고립계에서 총 entropy( 무질서도 ) 의 변화는 항상 증가하거나 일정하며 절대로 감소하지 않는다 .
에너지는 방향이 있다는 것이다 .
ΔS≥0부등호는 비 가역과정을 나타내고등호는 가역과정을 나타낸다 .
Basic Thermodynamics (3/9)
At Protein-Ligand Interactions
The Second Law of Thermodynamics
or
By defining change in “Entropy” as
or
0
T
Q0
T
Qd reversible
T
QS
0 gsurroundinsystem SS 0dS
Basic Thermodynamics (4/9)
At Protein-Ligand Interactions대부분의 protein-ligand interactions
At constant temperature & PressureOnly work is –PΔV
We can change this term to ΔH, then
0
T
VPES systemsystem
VPE
0
T
HS 0 HST
Basic Thermodynamics (5/9)
With the definition of (Gibbs) 'Free Energy' as
ΔG < 0 : spontaneous change
ΔG = 0 : Equilibrium
STHG
STHKRTG b ln
Basic Thermodynamics (6/9)
ΔH 의 효용성
Direct measurement of heat of reaction
No ΔPV-work is the same as ΔH
PVHE
HE
Basic Thermodynamics (7/9)
Indirect measureUtilizes a simplified relationshipThe Van't Hoff Equation
Gibbs Free Energy Equation
At steady state, at which ΔG=0, then
Basic Thermodynamics (8/9)
LPPL
K
PLLP
eq
LP
PLln0 RTGG
LP
PLln0 RTG
Gibbs Free Energy Equation
This is an integrated form of theVan't Hoff Equation
Basic Thermodynamics (9/9)
dd
eq KRTK
RTKRTG ln1
lnln0
R
S
TR
HKd
00 1ln
2
0ln
RT
H
dT
Kd eq
Van't Hoff equation(1/2)
평형 상수의 자연로그와 온도의 역수 값에 대한그래프는 직선을 그린다 .이 직선의 기울기는 엔탈피의 변화량을기체상수로 나누어준 값의 음의 값이다 .절편값은 엔트로피의 변화량을 기체상수로나누어준 값이다 .이 식을 미분형태로 표현한 것이Van't Hoff Equation 이다 .
R
S
TR
HKd
00 1ln
Van't Hoff equation(2/2)온도 변화에 따른 평형상수 (K) 의 변화 비를엔탈피 변화를 이용하여 표현
2
0ln
RT
H
dT
Kd eq
Application
Application실험 data 는 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 parameters 를 측정
생체 물질의 interaction
Drug 나 Enzyme 에 관련해서 직접적으로
축적된 3-D protein structures 의 이해
여러 가지 결합 상황을 예측 , design 가능
Weak forces 로 이루어지는 protein-ligand
interaction 을 분석 , 추정
Summary
Advantages / Disadvantages
Advantages
Immobilization or
labeling 이 필요 없다 .
다양한 적용 범위
Kd, ΔH 측정
다른 온도와 pH 에서
가능
Disadvantages
Enormous amounts
of binding partner
Only medium affin-
ity
많은 membrane
proteins 에 제약
비싼 가격
SummaryThermodynamic parameters
Characterization and understanding of chemi-cal
reaction
Protein-ligand 영역으로의 확장Drug-discovery 등의 다양한 영역에 실용적
이전 van't Hoff technique 에서 발전Modern, automated, high-sensitivity calorime-try equipment
Proteinomics 관심 대상Biomolecules 의 folding 이나 ligands 의 결합