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CALORIMETRIAstrumenti ed applicazioni

Università di Parma, 9 novembre 2010R. Pepi – TA Instruments

Thermal Analysis

”Thermal analysis refers to a group of techniquesin which a physical property of a substance is measured as a function of temperature whilst the substance is subjected to an imposed temperature program under controlled atmosphere”Examples: DSC, TGA, TMA,

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Calorimetry

”Calorimetry refers to those measuring techniques that are used for direct determination of rate of heat production, heat and heat capacity as function of temperature and time” Examples: DSC, TAM, ITC

TA vs. Calorimetry

Thermal Analysis Calorimetry

Scanning, Temperature-Induced Processes

Scanning or Isothermal

DSCTAMTMA

TGA

DMAReaction Calorimetry

Solution Calorimetry

ITC

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Calorimetry Features

• All kinds of processes result in either heat production or heat absorption: Chemical, Physical and Biological

• Non-specific

• Direct and continous

Reaction rate ó Heat production rate

Reaction rate àKinetic Information

Concentration àAnalytical Information

)(

)(

cfkHP

HdtdCP

cfkdtdC

⋅⋅∆=

∆=

⋅=

Enthalpy àThermodynamic Information

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Heatflow vs. Time

Shows how the reaction rate varies with time.

P = ΔH dC/dt

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f(t, P, Q)

Hea

t flo

w (µ

W/g

)

Time (d)

Energy vs.Time

Shows how the extent of reaction varies with time.

P dt = ΔH dC

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Ener

gy (J

/g)

Time (d)

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Heatflow vs. Energy

“Shows how the reaction rate varies with the extent of reaction”

(ex. 1 order → Q proportional to Pwith rate const., k, as proportionality constant)

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Calorimetry Instruments• TAM III

– Isothermal Calorimeter (±0.00005°C)– Versatile, Modular System

§ Stability/Compatibility§ Perfusion§ Isothermal Titration Calorimetry§ Solution Calorimetry

• Nano-ITC– Dedicated Isothermal Titration Calorimeter– (Protein) Binding– (Protein) Interactions

• Nano-DSC– Dedicated Scanning Calorimeter– (Protein) Structure/Stability

• MC-DSC

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Calorimetric Range

-12 -2

-3 -1

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4-12 -10 -8 -6 -4

-11 -9 -7 -5

1 pW

1 nW

1 µW

1 mW

1 W

General Features of TAM• Thermostat

– Controls the temperature• Calorimeters

– Contains the heat detectors• Sample handling systems

– Contains the sample• Auxiliary equipment

– Equipment used to control the experimental conditions such as relative humidity, oxygen concentration.

• Software

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Heat exchange by Peltier coolers

Circulation Pump

Oil expansion tank

ComputerThermostat

Power Supply

Keyboard

Calorimeters

Monitor

TAM: an Integrated System

Twin System(used in TAM)

SΦSample holder

Sk

Surrounding(heat sink)

RΦSample holder

Rk

Surrounding(heat sink)

Sample cell (S) Reference cell (R)

SCsP

∆T=(Ts-To)-(TR-To)=Ts-TR

ST

oT

RCrP RT

To

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TS

To

P

dtdTC

TS

Heat Balance Equation

Rate of Heat Accumulation or depletion

Rate of Heat Production

Heat flow

Φ

P

dtdTCΦ= +

= +

General Heat Balance Equation

High Sample Throughput

• TAM is a multichannel microcalorimetric system offering up to 48 experiments to be performed.

• TAM is ideal for research purposes as well as for large scale screening

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TAM Calorimeter Features

• All kinds of processes; Chemical, Physical and Biological

• Non-specific• Non-destructive (sometime)• Not dependent on the physical shape of the sample• No need for sample preparation• Direct and continous

4-ml Nanocalorimeter

Foil Heaters for Calibration

Heat Sink Partition wall

Sample Ampoule

holder

Reference Ampoule

holder

Twin System

Thermoelectric Modules (Seebeck modules)

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Minicalorimeter (4 mL)

Outer Steel Cylinder

Reference Ampoule

holder

Thermopiles

Sample Ampoule

holder

Heat Sink

Sample Handling Systems

• Closed or sealed (static) Ampoules • Open ampoules - Micro Reaction System• Micro Solution Ampoule

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TAM Stability Testing

Time

Hea

t Flo

wex

o

Least stable

Most stableX

X

X

Flow calorimetry: Leukemia (T-lymphoma) cells exposed to the anti-cancer drug methotrexate. The final drug concentrations were (a) 0, (b) 0.2, (c) 0.5, (d) 1.0, (e) 2.0, (f) 4.0µM (ref 6).

Bermudez, Backman and Schon., Cell. Biophys. 20, 111-123, (1992).

Drug Efficacy

abc

def

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Response of Streptococcus Mutants

Morgan, Beezer, Mitchell and Bunch, J. Appl. Microbiol. 90, 53-58, (2001).

Flow calorimetry: in Absence and in Presence of an Antimicrobial Agent

Oxidation of Meclofenoxate Hydrochloride

Tomoko Otsuka, Sumie Yoshioka, Yukio Aso and Tadao Terao, National Institute of Hygienic Sciences, Tokyo, Japan, Chem. Pharm. Bull. 42(1) 1994

50°C

30°C

40°C

23°C

Hea

t Flo

w (µ

W)

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Specifically designed to measure molecular interactions and reaction kinetics.

Capable of thermal measurements over a wide variety of solution conditions and temperatures.

In-solution universal detector for maximum data collection in a single exper iment.

Nano-ITC

ITC Applications

• Molecular Binding Studies– Quick and accurate affinities– Structure-function relationships– Affinity and mechanism of action screening– Specific vs. non-specific binding– Quality and process control– Protein engineering assessment– Validate virtual models– Drug resistance and adaptation evaluation

• Reaction Kinetics– KM, Vmax, kcat

– Enzyme Inhibition

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Nano ITC SchematicA. Heating and cooling TEDB. ThermistorC. Cylindrical cellsD. Constant coolingE. Power compensation heatersF. Thermal shieldG. ThermosensorH. Syringe stepper motorI. Temperature control block J. Temperature measurement K. Power compensationL. Signal amplifier M. Feedback control algorithmN. Temperature control algorithm O. Syringe control algorithm

Nano ITC Cell Design

Conical tops and bottoms promote consistent bubble free filling, uniform stirring and easy cleaning. Cylindrical cells are

made of chemically inert 99.999% pure gold.

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Twisted stirrer paddle

Threaded syringe mount

Embedded linear actuator

Spring loaded electronic connections for wire free operation

Nano ITC Syringe & Stirrer Design

Isothermal Titration Calorimetry

Typical ITC Data

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Hea

t R

ate

/ µW

ITC Strength:• Direct measure of Enthalpy, ∆H• Determine Binding affinity, Ka

• Determine Stoichiometry, n• Determine Entropy, ∆S

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Incremental titration, medium binding

• 2’-CMP (100 µL, 1.6 mM) titrated into RNase A (1.0 mL, 80 µM)• 20, 5 µL injections at 25 oC

• n = 1• Ka = 1 x 106 M-1

• Enthalpy of binding: -65 KJ/mol

The shape of the binding curve determines the accuracy of Ka. Need curvature.

What if Ka is outside 103 – 109 M-1?

• Regular (incremental) ITC experiment: 20 -30 data points, 1.5 - 2 hours

• Continuous ITC experiment: a thousand data points → more precise fit of data curve → more accurate Ka, n

• Continuously inject ligand (0.05 - 0.15 µL/s)

• 20 minute experiment

• No hardware or software modifications

• However, binding must be instantaneous

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Continuous titrationBaCl2 titrated into 18-crown-6

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Raw DataFit

n = 1.01Ka = 5.97 x 103 M-1

∆H = -31.4 kJ mol-1

2'-CMP titrated into RNase A

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Raw DataFit

n = 1.04Ka = 1.12 x 106 M-1

∆H = -73.4 kJ mol-1

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• Critical micelle concentration (CMC) is the concentration at which detergents aggregate to form micelles.

• Titrate concentrated detergent suspension (micelles) into buffer.

• Initially micelles dissociate in sample cell. At CMC, detergent in the sample cell aggregate. Midpoint of the inflection is the CMC.

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Critical micelle concentration

NanoITC Advantages

• Precision measurement of (biological) interactions - Heat is a universal detector of all processes

• NanoITC is the most versatile microcalorimetry instrument available for characterization and analysis of interactions of (biological) molecules

• In-solution; Native materials - no need to label or immobilize

• NanoITC generates full thermodynamic profile

• NanoITC is necessary tool for precision measurements of structure, function, and biomolecular interactions

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Specifically designed to determine thermal stability and heat capaci ty of proteins and other macromolecules in dilute solution. Unsurpassed assay precision enables maximum data collection in a single exper iment.

Nano-DSC

Nano DSC Applications

• Investigate Protein/Domain structure

• Determine thermal transition(‘melting’) temperatures

• Measure ΔH of denaturation

• Measure reversibility of thermal processes

• Measure ΔCp of the unfolding process

• Pre-formulation stability studies

• Evaluate relative stability of bioengineered proteins

• Evaluation of high affinity binding (up to 1020 M-1)

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DSC Block Diagram

• Cell Construction; Inert to biomaterials 99.99% Platinum

• Small Sample Volume(0.33 mL)

• Attenuates or delays onset of aggregation until after protein has unfolded

• Easy-to-fill and clean design

Continuous Capillary

Cylindrical

• Cell Construction; Inert to biomaterials 99.999% Gold

• Small Sample Volume(0.33 mL)

• Preferable for high temperature option

• Competitive “Non-Capillary” option

DSC Cell Design

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Differential Scanning Calorimetry

30 40 50 60 70 80 90

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Cp

(kca

l/mol

e/o C

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∆Cp}

TM

∆H

Typical DSC Data

Nano DSC Cell Configuration

Continuous capillary cells delay / inhibit aggregation and precipitation of

proteins during scansAggregation/Precipitation Characteristics

• Difficult to control• Expt parameters important• Kinetic event• Distortion of peaks – inaccurate data

Typical Non-capillary CellAggregation/Precipitation DSC Thermogram

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• If a ligand binds to a protein, the Tm of the protein will increase. Generally, the more bound ligand there is, or the tighter it binds, the more Tm increases.

• Can determine binding constant at Tm. Not ideal

• But, useful if very slow or very tight binding, or organic solvents necessary.

Ligand binding

• Perfectly valid if comparing relative binding of ligands to same protein• DSC is a quick way of screening whether two molecules interact.

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ss C

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ol-1

K-1

0.05 mM

0.075 mM

0.15 mM

0.3 mM0.75 mM

1 mM

1.25 mM

1.5 mM

0 mM

• About 30% of proteins are associated with membranes. Membranes are hydrophilic outside, hydrophobic inside.

• Membrane proteins are mostly hydrophobic, difficult to work with, very difficult to purify

• Does a protein bind to a membrane? Easy to tell by DSC

• Specific changes in lipid thermogram indicates how protein interacts with membrane (on surface, or internal penetration)

• Powerful! Most techniques are affected by cloudiness, light scattering. Not DSC!

Membrane protein/membrane interaction

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• Membrane proteins are very difficult to purify. Micrograms of protein can represent weeks of work. Require detergents for solubilization

• Using the Nano-DSC, obtained these high-quality scans using 20 µg (0.3 picomoles) of a 70,000 Da complex consisting of 3 membrane proteins

• Thermogram very well fit by 3 transitions

• Partial chemical derivatization and stablilization of complex easily verified by DSC

Membrane protein structure

• Pressure perturbation: the heat change in a biopolymer sample caused by a pressure jump.

• Heat corresponds to the work done by the pressure to create a volume change. Allows calculation of the coefficient of thermal expansion of the biopolymer, which is correlated with hydration of the biopolymer.

• Volume change can also be correlated with tightness of packing of protein interior (chymotrypsinogen is more hydrophobic than ribonuclease).

• Nano-DSC can alter pressure quickly and smoothly, in 3 modes.

• A potentially powerful technique, but very limited interpretable data available.

Pressure perturbation

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Summary and outlook

• The intricate structures of biopolymers are responsible for their activity. The structures are held together by many weak interactions. Proteins are only marginally stable.

• Understanding the thermodynamics driving biopolymer structure/function lets you design better proteins, design better ligands, understand how biopolymers fold, how they interact with their environment…

• Calorimetry is most direct, versatile and powerful approach for understanding the forces controlling protein structure and function.

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