itc (isothermal titration calorimetry) 황정현. contents introduction itc technology principle...

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