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)- calorimeter 열량계

Thermometric Titration A number of instrumental titration techniques Accurate and precise without a subjective interpretation 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 : 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 concentrations of the interacting components as molar heats(y-axis) and molar ratio(x-axis).

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 or By defining change in “Entropy” as

Basic Thermodynamics (5/9) At Protein-Ligand Interactions 대부분의 protein-ligand interactions At constant temperature & Pressure Only work is –PΔV We can change this term to ΔH, then

Basic Thermodynamics (6/9) With the definition of (Gibbs) 'Free Energy' as ΔG＜ 0 : spontaneous change ΔG = 0 : Equilibrium

Basic Thermodynamics (7/9) Direct measurement of heat of reaction No ΔPV-work is the same as ΔH

Basic Thermodynamics (8/9) Indirect measure Utilizes a simplified relationship The Van't Hoff Equation Gibbs Free Energy Equation At steady state, at which ΔG=0, then

Basic Thermodynamics (9/9) Gibbs Free Energy Equation This is an integrated form of the Van't Hoff Equation

Van't Hoff equation(1/2) 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

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 축적된 3-D protein structures의 이해 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 Immobilization or labeling이 필요 없다. 다양한 적용 범위 Kd, ΔH 측정 다른 온도와 pH에서 가능 Disadvantages Enormous amounts of binding partner Only medium affinity 많은 membrane proteins에 제약 비싼 가격

Summary Thermodynamic parameters Protein-ligand 영역으로의 확장 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