itc (isothermal titration calorimetry )
DESCRIPTION
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 - PowerPoint PPT PresentationTRANSCRIPT
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ITC(Isothermal Titration Calorimetry)
황정현
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ContentsIntroduction
ITC technology
Principle
Application
Data analysis
Summary
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Introduction
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IntroductionIsothermal Titration Calorimetry
Iso- : 같은 , 동 ( 同 )-, 등 ( 等 )-
Thermal : ‘ 열’의
Titration : 적정
Calorimetry : 열량 측정
calorimeter 열량계
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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)
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ITC Technology
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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)
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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
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Differential Scanning Calorimeter (2/4)
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Differential Scanning Calorimeter (3/4)
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Differential Scanning Calorimeter (4/4)
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DSC thermogram (1/2)Glass transition temperature(Tg)
Melting temperature(Tm)
Crystallization temperature(Tc)
결정화 시간 , 순도 , 산화 , 분해
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DSC thermogram (2/2)
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Isothermal Titration Calorimeter (1/6)Biomolecular
interactions 에 관한
연구
protein-ligandprotein-DNAAntibody-antigenHormone-receptor
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Isothermal Titration Calorimeter (2/6)모든 binding
parameter 를 측정
Binding 이 나타날 때Heat is taken up
- Absorbed- endothermic
Heat is evolved- Released- exothermic
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Isothermal Titration Calorimeter (3/6)
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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.
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Isothermal Titration Calorimeter (5/6)The reference cell is
electrical heated preset steady temperature.
The temperature difference between reference and sample cell is measured.
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Isothermal Titration Calorimeter (6/6)
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ITC – Before Titration
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Titration Begins : First Injection
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Return to Baseline
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Second Injection
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Second Return to Baseline
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Injections Continue
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Injections Continue
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End of Titration
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ITC – Fitting the Data (1/3)
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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).
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ITC – Fitting the Data (3/3)Fitting of this curve
gives the
parameters derived
in the text.
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Principle
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PrincipleBiological macromolecules 의 interaction
Molecular recognition 의 complexity and
diversity
Immune response, signal transduction cas-
cades, gene expression 등 중요 요인에
대한 관심과 적용
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연관변수를 측정하여 대상의 정체를 확인
n : Stoichiometry of the interactionKa : Association constantKd : Dissociation constantΔGb : Free energyΔHb : EnthalpyΔSb : EntropyΔCp : Heat capacity of binding
Basic Thermodynamics (1/9)
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At Protein-Ligand InteractionsThe First Law of Thermodynamics
열역학 제 1 법칙
ΔE=Q+WΔE represents the change in the energyQ the heat absorbed by the systemW the work done on the system
Basic Thermodynamics (2/9)
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At Protein-Ligand InteractionsThe Second Law of Thermodynamics
열역학 제 2 법칙
고립계에서 총 entropy( 무질서도 ) 의 변화는 항상 증가하거나 일정하며 절대로 감소하지 않는다 .
에너지는 방향이 있다는 것이다 .ΔS≥0
부등호는 비 가역과정을 나타내고등호는 가역과정을 나타낸다 .
Basic Thermodynamics (3/9)
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At Protein-Ligand InteractionsThe Second Law of Thermodynamics
or
By defining change in “Entropy” as
or
0
TQ 0
TQd reversible
TQS
0 gsurroundinsystem SS 0dS
Basic Thermodynamics (4/9)
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At Protein-Ligand Interactions대부분의 protein-ligand interactions
At constant temperature & PressureOnly work is –PΔV
We can change this term to ΔH, then
0
TVPE
S systemsystem
VPE
0
THS 0 HST
Basic Thermodynamics (5/9)
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With the definition of (Gibbs) 'Free Energy' as
ΔG < 0 : spontaneous change
ΔG = 0 : Equilibrium
STHG
STHKRTG b ln
Basic Thermodynamics (6/9)
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ΔH 의 효용성
Direct measurement of heat of reaction
No ΔPV-work is the same as ΔH
PVHE
HE
Basic Thermodynamics (7/9)
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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)
LPPLK
PLLP
eq
LPPLln0 RTGG
LPPLln0 RTG
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Gibbs Free Energy Equation
This is an integrated form of theVan't Hoff Equation
Basic Thermodynamics (9/9)
dd
eq KRTK
RTKRTG ln1lnln0
RS
TRHKd
00 1ln
2
0lnRTH
dTKd eq
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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 .
RS
TRHKd
00 1ln
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Van't Hoff equation(2/2)온도 변화에 따른 평형상수 (K) 의 변화 비를엔탈피 변화를 이용하여 표현
2
0lnRTH
dTKd eq
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Application
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Application실험 data 는 protein-ligand 연구정보를
참고하여 분석
MEDLINE search
ITC equipment suppliers
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MEDLINEMedical Literature Analysis and Retrievalsystem OnlineBibliographic database of life science andbiomedical informationMedicine, nursing, pharmacy, dentistry,health care, biology, biochemistry andmolecular evolutionSearchable via PubMed
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Data Analysis
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생물리학 연계성
Thermodynamic parameters 를 측정
생체 물질의 interactionDrug 나 Enzyme 에 관련해서 직접적으로
축적된 3-D protein structures 의 이해
여러 가지 결합 상황을 예측 , design 가능
Weak forces 로 이루어지는 protein-ligandinteraction 을 분석 , 추정
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Summary
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Advantages / DisadvantagesAdvantages
Immobilization orlabeling 이 필요 없다 .다양한 적용 범위
Kd, ΔH 측정
다른 온도와 pH 에서
가능
DisadvantagesEnormous amountsof binding partnerOnly medium affin-ity많은 membraneproteins 에 제약
비싼 가격
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SummaryThermodynamic parameters
Characterization and understanding of chemi-cal
reactionProtein-ligand 영역으로의 확장
Drug-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