MICROCALORIMETRY: ITC & DSC

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New Castle, DE USA

Lindon, UT USA

Saugus, MA USA

Hüllhorst, Germany

Shanghai, China

Beijing, China

Tokyo, Japan

Seoul, South Korea

Taipei, Taiwan

Bangalore, India

Sydney, Australia

Guangzhou, China

Eschborn, Germany

Wetzlar, Germany

Brussels, Belgium

Etten-Leur, Netherlands

Paris, France

Elstree, United Kingdom

Barcelona, Spain

Milano, Italy

Warsaw, Poland

Prague, Czech Republic

Sollentuna, Sweden

Copenhagen, Denmark

Chicago, IL USA

São Paulo, Brazil

Mexico City, Mexico

Montreal, Canada

MicrocalorimetryIsothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) are powerful analytical techniques for in-depth characterization of molecular binding

events and structural stability. Thermodynamic binding signatures not only reveal the strength of a binding event, but the specific or nonspecific driving forces involved.

Structural stability profiles from DSC reveal strengths and weaknesses in higher order structure and define the behavior of individual domains and their interactions. The

TA Instruments Affinity ITC, Nano ITC and Nano DSC provide the performance, reliability and ease-of-use required for the most demanding applications in drug discovery,

protein-protein interactions, structure-function characterization and more.

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Affinity ITC and ITC AutoISOTHERMAL TITRATION CALORIMETER

Affinity ITC Auto 3

The Affinity ITC and ITC Auto are designed for the most challenging life science laboratory environments that require high sensitivity, high productivity and the most advanced ITC technologies. The Affinity ITC brings advanced engineering to all critical aspects of the measurement ensuring the highest quality ITC data.

Features: •AccuShot™deliversthetitranttotherightlocationforthebestmixing

•FlexSpin™providesinnovativeslowspeedstirring,efficientmixingandhighest

sensitivity

•Fullyautomated,user-selectablesystemcleaningroutineseliminaterun-to-run

contamination

•IntelligentHardwarePositioningforprecisereliableinjections

•Solid-stateactiveheatingandcoolingfortrueisothermaltemperaturecontrol

•Choiceofstandardvolume(1.0mL)orlowvolumecells(190µL)

•Industryproven96-well,temperature-controlledliquidhandlingautosampler.

Autosampler can be included with initial purchase or added at a later date

•PowerfulITCRunandNanoAnalyzeforthemostcomprehensivesuiteoftoolsfor

methodoptimization,modelfitting,batchanalysis,graphinganddataexport.

TA Instruments has perfected what others have attempted. The Affinity ITC is a powerful

toolformeasuringawidevarietyofmolecularinteractions.Itprovidesbothinexperienced

and advanced ITC users the highest confidence in generating superior ITC data.

The Affinity ITC cell is optimized in shape, material, and volume to provide the greatest measurement accuracy over the widest range of sample chemistries.

Choice of Cell Volumes:The Affinity ITC features two fixed-in-place calorimetric cells: a sample cell where

injectionstakeplaceandamatchingreferencecell. Twocellvolumesareavailable:

1.0mL(StandardVolume)and190µL(LowVolume).Automationisavailableineither

configuration.

Selection of cell volume depends on the range of binding constants to be measured (Kd:

mMtolownM)andtheavailabilityofsample.TAInstruments’experiencedapplication

teams can recommend the best instrument configuration for your specific measurement

requirements.

Cylindrical Cell Geometry:Thecylindricalcellgeometrymaximizesstirringefficiency,eliminatesdeadzonesand

entrapped air bubbles which are common in competitive designs.

Cell Composition:Tomaximizemeasurementaccuracyand response, theAffinity ITCStandardVolume

configurationhascellsconstructedofeither24k99.999%Gold(Au0)orHastelloy.The

LowvolumeconfigurationisonlyavailablewiththeGold(Au0)cells.Theinertchemical

propertiesofGold,itshighthermalconductivityanditsabilitytobecleanedwithstrong

acids and bases make it the preferred choice for ultrasensitive ITC instruments.

Affinity ITC TECHNOLOGY

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StandardVolume(1.0mL)

LowVolume(190µL)

Technology 5

Solid-State Temperature Control & Power Compensation Operation. The Affinity ITC utilizes multiple solid-state thermo-electric elements for active heating and cooling of the sample and reference cells.

Advantages of active heating and cooling: •Fasterheatingandcoolingbetweentemperaturesetpoints

•Rapidequilibrationattemperaturesetpoint

•ActivetemperaturecontroleliminatesdriftonlongITCexperiments

The absorption or evolution of heat as a result of a binding reaction is detected by the

thermoelectricelementsafterwhichheaterpowerisadjustedtomaintainthetemperature

difference between the sample and reference cell at zero. The combination of power

compensation and thermoelectric temperature control ensures the fastest response and

highest resolution for ITC.

New FlexSpin technology dramatically improves one of the most important aspects of ITC experiments, mixing.

Features: •Revolutionarynewpaddleshapeanditsseparationfrominjectionsystemresultsin

bettermixing,sharperpeaksandfasterreturntobaseline

•Moreefficientmixingandprecisedeliveryoftitrantreducespeakwidthupto50%

•Improvedsampledeliverysystemdecreasesequilibrationtimesby40%

•Slowerstirspeeds(10Xslowerthancompetitiveinstruments)forhighestsensitivity

while protecting delicate structures

•Inconjunctionwithcylindricalcellshape,eliminatesdeadzones

•Suspendedonaflexiblesupport,eliminatingthepossibilityofdamagedueto

misalignment

Affinity ITC TECHNOLOGY

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Titrant Delivery

FlexSpin Stirrer

The precision and the location of the titrant delivery are critical to obtaining the highest quality ITC data. The AccuShot injection system has been completely redesigned to optimize these factors. AccuShot delivers the right amount of titrant in the right location, every time.

Features: •Injectionsystemseparatefromstirringmechanism

•Syringeneedlepositionedtodelivertitrantatthetopofstirringpaddleforbetter

mixingandsharperpeaks

•Highprecisionsteppermotorforthemostaccuratedeliveryofa0.01µLto250µL

injection

• Improvedsampledeliverysystemdecreasesequilibrationtimesby40%

•Smalldiametercannulaminimizes1stinjectiondiffusion

•Singlesyringeforallinjectionvolumesandexperimentdesigns

•Quick,easysyringereplacement

•Easytitrantloadingwithoutinjectionsyringeremoval

•Fullyautomatedinternalandexternalcleaningofinjectioncannula

Technology 7

Affinity ITC Auto TECHNOLOGY

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What others have attempted TA Instruments has perfected.By combining automation and liquid handling technology prevalent in today’s life science laboratories with the high performance Affinity ITC, TA has created the most powerful platform for automated, high throughput ITC experiments.

Features and Benefits of the Affinity ITC Autosampler: •HPLCgradeliquidhandling

•Smallbenchtopfootprinttooptimizespace

•Samplesconfiguredintwo(2)96-wellplates

•Userselectabletemperaturecontrol(ambientdownto4°C)

•Fullsamplepathwaycleaningcapability

•Batchfileprocessingforrapidanalysisandreporting

•Samplethroughput:From10to50perdaydependingonexperimentalconditions

•OptimizationtoolinNanoAnalyzeguidesuserstoproperexperimentalparameters

andmaximizesthroughput

9HardwarePositioning

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Previous attempts at ITC automation commonly experience misalignment, broken and bent syringes and overall reliability issues. Intelligent Hardware Positioning eliminates these issues and ensures accurate and reliable placement of automated hardware on the Affinity ITC.

Features: • Alignmenttabsensureperfectpositioningofthecellloadingneedle,stirring

mechanism,andtitrantinjectioncannulaintothesamplecell

• Self-aligningarmsarepartofaproprietarydesignthatprovidessafe,errorfree

positioning

The Intelligent Hardware Positioninggives users a new level of confidence for safe,

reliable, unattended, continuous operation of the Affinity ITC.

Affinity ITC Auto INTELLIGENTHARDWAREPOSITIONING

Autosampler Cleaning 11

The automated cleaning system engineered into the Affinity ITC Auto instrument ensures that the entire system is cleaned between sample titrations. This completely eliminates cross-contamination from one run to another.

Cleaning Features: •Dedicatedwash/rinsestationsforstirring/injectionsyringesandcellcleaning/filling

•User-programmablecleaningroutinescanbesavedandretrieved

•Fullyautomatedcleaningoftheinternalandexternalsurfacesofkeycomponents,

including autosampler transfer syringe and needle

•Fiveuser-selectablecleaningsolutions

•Completesamplepathwaycleaning

Autosampler Stream Selection Valve

Dedicated Wash Station

flush

Affinity ITC Auto Affinity ITC

Injection/Stirring cleaning Fullyautomated Fullyautomated

Cell cleaning Fullyautomated Cleaning tool

Cell filling cleaning Fullyautomated Manual

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Affinity ITC Auto PERFORMANCEDATA

TwelveRNAse-2’CMPTitrations

Affinity ITC Auto Reproducibility

The Affinity ITC Auto provides unmatched sample reproducibility with the highest

sensitivity and reliability. The figure shows twelve titrations performed with complete

system cleaning performed between each titration. Data plots are offset for display

purposes.

13PerformanceData

Affinity ITC Auto Cleaning Efficiency

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Two Buffer titrations performed after RNase-2'CMP titrations

Two RNase-2'CMP titrations

Two Buffer titrations performed before RNase-2'CMP titrations

Complete system cleaning is user-programmable with Affinity ITC Auto instrument

control software. Choosing from five (5) solvent ports ensures the entire sample path

is clean. The buffer titrations before and after the protein-ligand titrations provide the

highest confidence in the cleaning protocol for the Affinity ITC Auto.

Nano ITCISOTHERMAL TITRATION CALORIMETER

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15Nano ITC

The NANO ITC features many of the high performance technologies found in the Affinity ITC. It is a versatile, high-sensitivity, cost-effective isothermal titration calorimeter that can easily outperform competitive systems in a wide range of applications.

Features: •ChoiceofStandardVolume(1.0mL)orLowVolume(190µL) cells

•Solid-stateactiveheatingandcoolingfortrueisothermaltemperaturecontrol

•Highprecisioninjectionburetforaccuratetitrantdelivery

•Uniqueremovableinjectionsyringeforfastreliableloadingandcleaning

•PowerfulITCRunandNanoAnalyzeforthemostcomprehensivesuiteoftoolsfor

methodoptimization,modelfitting,batchanalysis,graphinganddataexport

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Nano ITCTECHNOLOGY

StandardVolume(1.0mL)

LowVolume(190µL)

The Nano ITC cell is optimized in shape, material, and volume to provide the greatest measurement accuracy over the widest range of sample chemistries.

Choice of Cell Volumes:TheNanoITCfeaturestwofixed-in-placecalorimetriccells:asamplecellwhereinjections

take place and amatching reference cell. Two cell volumes are available: 1.0mL

(StandardVolume)and190µL(LowVolume).

Selection of cell volume depends on the range of binding constants to be measured (Kd:

mMtolownM)andtheavailabilityofsample.TAInstruments’experiencedapplication

teams can recommend the best instrument configuration for your specific measurement

requirements.

Cylindrical Cell Geometry:Thecylindricalcellgeometrymaximizesstirringefficiency,eliminatesdeadzonesand

entrapped air bubbles which are common in competitive designs.

Cell Composition:Tomaximizemeasurementaccuracyand response, theNano ITC StandardVolume

configurationhascellsconstructedofeither24k99.999%Gold(Au0)orHastelloy.The

LowvolumeconfigurationisonlyavailablewiththeGold(Au0)cells.Theinertchemical

propertiesofGold,itshighthermalconductivityanditsabilitytobecleanedwithstrong

acids and bases make it the preferred choice for ultrasensitive ITC instruments.

17Technology

Thermoelectric Devices

Sample/Reference Cells

Thermal Shield

Cell Access Tubes

The Nano ITC utilizes multiple solid-state thermoelectric elements for active heating and cooling of the sample and reference cells. A unique removable buret and injection syringe ensures easy sample loading and accurate sample delivery.

Accurate Temperature Control with Active Heating and Cooling: •Fasterheatingandcoolingbetweentemperaturesetpoints

•Rapidequilibrationattemperaturesetpoint

•ActivetemperaturecontroleliminatesdriftonlongITCexperiments

The absorption or evolution of heat as a result of a binding reaction is detected by the

thermoelectricelementsafterwhichheaterpowerisadjustedtomaintainthetemperature

difference between the sample and reference cell at zero. The combination of power

compensation and thermoelectric temperature control ensures the fastest response and

highest resolution for ITC.

Unique Injection Buret and Removable Injection Syringe: •Accuratecontrolofthetitrantvolumedeliveryanduser-selectablestirspeedis

accomplished with a unique, easily removed buret

•Removableinjectionsyringesallowsthoroughcleaningandeasysampleloading.

•Partialsyringefillsforshorttitrationsareuser-programmableintheinstrument

control software

ITC TheoryAll molecular interactions have a unique thermodynamic signature that is char-

acterized by

•Bindingconstant(Ka)

•Enthalpy(ΔH)

•Entropy(ΔS)

•Stoichiometry(n)

ITC is a label-free direct measurement of the heat evolved or absorbed during a binding

reaction.

Thedatafromatypicalbindingexperimentisshowntotheright.Theintegratedareas

undereachinjectionpeakareplottedagainstthemolarratiooftheactivespecies.An

independentbindingmodelisfittothisdatatodirectlydeterminetheenthalpy(ΔH),

binding constant (Ka) and stoichiometry (n). Ka represents the association binding

constant. The dissociation binding constant, Kd,isdefinedas1/Ka.

GibbsFreeEnergyiscalculateddirectlyfromthebindingconstant,Ka

ΔG 0 = -RT LnKa

Thechangeinentropy(ΔS)termisthencalculateddirectly

ΔG 0=ΔH - TΔS

ITC is a powerful analytical technique that does not require labeling or immobilization

and is considered the most sensitive, accurate assay method for optimizing laboratory

productivity in life science applications, such as drug discovery and validation, molecular

variant comparisons and protein-protein interactions.

The Power of ITCTHEORY

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Thermodynamics Reveals Nature of Binding

DG0 Total Binding Affinity

DHHydrogen-bond formation and van

der Waals interactions

DSDesolvation and

Conformational effects

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19Applications

Enzyme Kinetics Using ITC In addition to binding, an isothermal titration calorimeter is also a powerful tool to perform

enzyme kinetics analysis. The substrate concentration-dependent heat flow of enzymatic

reactions may be used for kinetics analysis and the determination of Michaelis-Menten

reactionparameters:

Benefits:

•Nolabeling,immobilizationormodificationrequired

•Nolimitonsolutionturbidity

•Idealforcharacterizationofnovelenzyme-substratereactions

•Continuousassay,noneedtoquenchthereaction

The rate of heat flow and reaction enthalpy are easily and accurately determined using

themultipleinjectionmethod(topfigure).Michaelis-MentenandLineweaver-Burkplots

(bottom figure) are used to fit the data from the top figure to determine reaction kinetics

parameters.

4.03.53.02.52.01.51.00.5

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[Substrate] (µM)

Vmax

Km

Model Variable ValueMichaelis-Menten Vmax (µM/s) 3.856 Kcat (s-1) 104220 Km (µM) 17959 [E0] (µM) 3.700e-5 Fit StdDev (µM) 1.615e-2

Lineweaver-Burk Vmax (µM/s) 9.595 Kcat (s-1) 259336 Km (µM) 78495 [E0] (µM) 3.700e-5 Fit StdDev (µM) 6.082e-4

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Raw Heat RateHeat Rate after injectionUser-excluded PointsPre-injection Heat Rate

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ITCAPPLICATIONS

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Nano ITC Low VolumeTotal Titration Time: 2500 sec

Nano ITC Standard VolumeTotal Titration Time: 6500 sec

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Low and Standard Volume Comparison ThesensitivityoftheNanoITCLowVolumeensuresthatwithlesssampletheinstrument

will generate accurate and reproducible results in a shorter overall titration time. The

StandardVolume and LowVolumeNano ITC instruments provide the flexibility and

sensitivityforperformingawidevarietyofITCexperiments.

Nano ITC Low Volume: •Sample Cell = KHCO3;0.36mM

•InjectionSyringe=HCl;4.2mM

•Injectionvolume=1.4μL

• Injectioninterval=175sec

•provides the highest sensitivity

•canproduceshortesttitrationtimes

• isidealformaximizingthedatawith

minimum sample consumption

Nano ITC Standard Volume: •Sample Cell = KHCO3;0.36mM

•InjectionSyringe=HCl;5.6mM

•Injectionvolume=5μL

• Injectioninterval=300sec

•allowsmoresamplemasstobeloaded

•produceshighqualitydatawhenthe

molecular interactions are high affinity and

yield low heat values

Applications

Characterizing Binding Interactions by ITCAll binding events are accompanied by the evolution or absorption of heat (a change

inenthalpy,ΔH). Inasingle ITCexperimenta full thermodynamiccharacterizationof

the binding reactions can be obtained.With the appropriate experimental design,

fundamental information about the molecular interactions driving the process, as well

as the stoichiometry of binding (n) and the binding constant (Kd) is generated. The first

figureshowsatypicalincrementaltitration(20,5μLinjections)ofaninhibitor,2’-CMP,

titratedintoRNaseA;n=0.99,Kd=5.92x10-7 M,andΔH=-64.4kJmol-1. The second figure

showsthesameexperiment,plottingtheindividualintegratedpeakareasvstheratio

of the two binding molecules. As the binding sites become saturated, the amount of

heatproducedwithindividualinjectionsdecreases.Theresultingtitrationcurvereveals

valuableinformationontheenthalpy(ΔH),entropy(ΔS)andoverallGibbsfreeenergy

(ΔG0) of the reaction taking place in the calorimeter. ITC is a powerful analytical tool and

considered the most sensitive assay technique for characterizing the fundamental driving

forces of molecular binding reactions.

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Kd = 5.92x10-7 M∆H = -64.4 kJ mol-1

n = 0.99

21

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Protein InteractionsWhen two proteins interact and bind, conformational changes in the proteins, and

rearrangement of the solvent in the vicinity of the binding site, result in the absorption

orgenerationofheat.QuantificationofthisreactionheatbyITCprovidesacomplete

thermodynamic description of the binding interaction, the stoichiometry of binding, and

the association constant. This figure contains the titration data of porcine pancreatic

trypsin into soybean trypsin inhibitor using a Nano ITC. Twenty, 5 µL aliquots of ligand were

titratedintothesamplecellwhilethetemperatureofthesystemwasmaintainedat25°C.

Toppanel:Thesignal(heat)producedfollowingeachadditionofproteintotheinhibitor.

Bottompanel:Integrationoftheheatsoverthetimecourseoftheexperiment;theµJin

each peak are plotted against the mole ratio of the titrant to inhibitor.

ITCAPPLICATIONS

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[Trypsin] / [Soybean Trypsin Inhibitor]

Kd = 1.4 x 10-7 M∆H = 135 kJ mol-1

n = 1.15

Applications

Characterization of Enzyme KineticsEvery reaction generates or absorbs heat, so every reaction can in principle be studied

by calorimetry. In practice it has been shown that representative enzymes from every EC

classification can be analyzed kinetically using ITC. In addition, ITC analyses are rapid,

precise, nondestructive, compatible with both physiological and synthetic substrates, and

are as sensitive as spectroscopic techniques but do not require a spectroscopic label

or chemical tag. Importantly, ITC analyses of enzyme kinetics are also straightforward.

Thefigureshowsthehydrolysisofasingle10µLinjectionoftrypsinintoasolutionofBAEE

in the absence (blue) and presence (red) of benzamidine, a competitive inhibitor. The

area under both curves (representing the total heat output for complete conversion of

substrate to product) is the same either in the presence or absence of inhibitor, allowing

the KM and kcat of the reaction under both conditions to be calculated, as well as the

inhibition constant.

Continuous Single Injection Continuoussingleinjectiontitrationisanattractivealternativetothetraditionalincremental

titrationITCforsamplesexhibitingveryrapidbindingreactions.Thesecontinuousinjection

experimentscanbecompletedinlesstotaltimethannormallyrequiredforafullsetof

incremental titrations. This technique provides accurate determinations of stoichiometry

(n)andenthalpy(ΔH)forawiderangeofbindingconstants.Continuousinjectionand

incrementalinjectionexperimentscanbeperformedinboththeITCstandardvolume

and low volume instruments with no alterations in hardware or software supplied with the

instruments.

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Kd = 8.9 x10-7 M∆H = -73.4 kJ mol-1

n = 1.04

23

Nano DSC and DSC Auto DIFFERENTIALSCANNINGCALORIMETER

24

Nano DSC 25

The Nano DSC has the versatility and precision for char-acterizing molecular stability, determining high affinity ligand binding and deconvoluting multi-domain structures. There is no other DSC with the proprietary technologies, high performance or the sample throughput of the Nano DSC and Nano DSC Auto.

Features: •Highestsensitivity,lowestcellvolumeforunmatchedperformance

•Capillarycelldesignforanalysisofsamplesthattendtoaggregateorprecipitate

•Built-inprecisionpressurizingsystemmaintainsaccurate,constantpressureinthe

cells

•Solid-statethermoelectricelementsforaccuratetemperaturecontrolduringheating

and cooling scans

•UpgradeablewithindustryprovenHPLCgradeautosamplerforreliablehighsample

throughput

26

The Nano DSC is designed for ultra-sensitive measure of heat absorbed or released by dilute in-solution bio-molecules as they are heated or cooled. The capillary cell design, solid-state thermoelectric temperature control and easy cleaning ensure the highest sensitivity and data reproducibility for a wide variety of applications.

Features: •300µL active volume capillary cells for analyzing hydrophobic samples

•Easy,accuratesampleloadingwithlaboratorypipetteman

•Built-in,user-programmablepressurizationsystem(upto6atm)

•Flexibledataacquisitioninterfaceforeasyexperimentsetup

•NanoAnalyzesoftwareforaccuratemodelfittingandmulti-filebatchprocessing

Nano DSC TECHNOLOGY

Capillary Sample/Reference Cells

Top PlateThermoelectric Device

Thermal Shield

Pressure Ring

27Technology

The NanoDSC is a powerful thermal scanning instrument that utilizes a 300 µL capillary cell design and solid-state thermoelectric temperature control to provide unmatched performance.

Nano DSC Capillary Platinum Cells •Fixed-in-placecapillarycellsattenuateaggregationandprecipitation

•Platinumcellsareinertandcompatiblewithstrongacids,basesandprotein

cleaning enzymes

•300µL active cell volume minimizes sample consumption

•Samplecellloadingwithlaboratorypipettemaniseasyandensuresnotrappedair

bubbles

Nano DSC Solid-State Thermoelectric Temperature Control: •Accurate,reproducibletemperaturecontrolforhighestsensitivityinbothheating

and cooling scans and unmatched baseline reproducibility

• Innovative,user-programmablebuilt-inpressuresystemforcomplexanalysisof

water characteristics and molecule structure

•User-programmablescanratesforscanflexibilityandhighestconfidenceindata

analysis

28

The Nano DSC Autosampler enables true “start and walk away” capability without sacrificing either sensitivity or reliability. It is an industry-proven 96-well plate autosampler that stores and delivers samples to the DSC cells. User-programmable washing routines ensure no sample carry over and the 96-well format maximizes sample throughput.

Autosampler Features: • Industry-provenHPLCautosamplerreliability

•EasyconnectiontotheNanoDSCthroughautosamplerinterface

•Two(2)96-wellplatesstoresamplesattemperaturesdownto4°C

•Four(4)wash/rinsesolventportsontheautosamplerinterfaceareuser-programmable

•Two (2)exit ports enable thecollectionof sampleandmatchingbuffer/solvent

solutions from both the sample or reference cells

•AutosamplerisprogrammablethroughtheNanoDSCinstrumentoperatingsoftware

Nano DSC AUTOMATION

How much Protein is Required for a DSC Scan? Determining the thermodynamic parameters of a protein by differential scanning

calorimetry (DSC) using the Nano DSC requires about the same amount of protein as

surfaceplasmonresonanceorfluorescencestudies.BecauseoftheNanoDSC’sextreme

sensitivityandbaselinereproducibility,andthesamplecell’ssmallvolume(300µL),a

complete, interpretable, accurate scan can be obtained on essentially any protein of

interest. The sensitivity and accuracy of the Nano DSC is demonstrated by this data. Hen

eggwhitelysozyme(inpH4.0glycinebuffer)waspreparedatvariousconcentrations.As

little as 2 µg of lysozyme in the capillary cell is sufficient to provide quality data yielding

accurate values of all four thermodynamic parameters!

29Automation

55 60 65 70 75 80 85 90 95

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Nano DSC APPLICATIONS

Lysozyme

in cell (50µg)

Calorimetric Van’t Hoff

DH(kJmol-1) DS(kJK-1mol-1) Tm (˚C) DH(kJ mol-1)

400 512 1.46 78.0 515

100 512 1.46 78.0 509

50 517 1.47 77.9 513

25 513 1.46 77.8 513

10 515 1.47 78.0 515

5 490 1.40 78.0 510

2 503 1.43 77.8 499

30

Characterization of Protein StabilityAnalyzing the stability of a protein in dilute solution involves determining changes in the

partialmolarheatcapacityoftheproteinatconstantpressure(ΔCp).Thecontributionof

the protein to the calorimetrically measured heat capacity (its partial Cp) is determined

by subtracting a scan of a buffer blank from the sample data prior to analysis. Heating the

protein sample initially produces a slightly increasing baseline but as heating progresses,

heat is absorbed by the protein and causes it to thermally unfold over a temperature

range characteristic for that protein, giving rise to an endothermic peak. Once unfolding

is complete, heat absorption decreases and a new baseline is established. After

blank subtraction, the data can be analyzed to provide a complete thermodynamic

characterization of the unfolding process.

Characterization of Protein Structure DSCcanbeusedtocharacterizeboththespecificbindingofaligand(forexample,a

drugtoareceptorbindingsite),ornonspecificbinding(forexample,detergentsbinding

to hydrophobic patches on a protein surface). In some instances ligand binding, even

if to a specific receptor site, results in long-range protein structural rearrangements that

destabilize theentirecomplex.The figure showsDSCscansofCa2+ saturated bovine

a-lactalbuminatvariousprotein:Zn2+ ratiosscannedat1°C/min.Themidpointof the

thermalunfoldingoftheproteindecreasesfrom65°CintheabsenceofZn2+to35°Cat

aprotein:Zn2+ratioof1:70.Theenthalpyofunfoldingisalsodecreasedsubstantiallyby

highZn2+ concentrations.

Nano DSC APPLICATIONS

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-3

-4

-5

-6

-7

-840

baseline scans

barnase scans

50 60 70 80

He

at F

low

/µW

Temperature (˚C)

exo

30 40 50 60 70 80 90

No Zinc

Exc

ess

He

at C

ap

ac

ity

Temperature (˚C)

1:3.5

1:14

1:70

31Applications

Investigation of Protein-Ligand BindingDSC is a valuable tool for studying binding between a biological macromolecule and a

ligandsuchasanotherbiopolymeroradrug.UnlikeITC,DSCallowsthethermodynamics

that drive binding to be correlated with conformational changes in the macromolecule

caused by the binding reaction. DSC is particularly useful for characterizing very tight

or slow binding interactions. DSC also allows characterization of binding reactions that

areincompatiblewiththeorganicsolventrequirementsofsomeITCexperiments(i.e.,

whereligandsolubilityforanITCexperimentrequiresconcentrationsoforganicsolvent

not tolerated by the protein). The data shows DSC scans of RNase A bound with increasing

concentrationsof2’-CMP,showingthattheproteinisstabilizedbyhigherconcentrations

of the inhibitor.Essentially identicaldatawasobtained in thepresenceof5%DMSO,

verifying that organic solvents are compatible with the DSC technique.

Nano DSC Capillary Cell AdvantagesThisfigureshowstwoDSCscansofmatchedsamplesofhumanIgG1at0.5mg/mlin

physiological buffer. The data from the DSC with a “coin” shaped sample cell shows the

easilyrecognizableexothermicaggregation/precipitationeventatapprox89-90°C,while

the data collected on the Nano DSC with a capillary sample cell shows a stable post-

transition baseline that will enable complete and accurate determinations of transition

temperatures(Tm)andenthalpy(ΔH).

35 40 45 50 55 60 65 70 75 80 85

0 mM0.05 mM0.075 mM0.15 mM0.3 mM0.75 mM1 mM1.25 mM1.5 mMC

p (

Kca

l/K-

mo

l)

Temperature (˚C)

18

16

14

12

10

8

6

4

2

0

-2

40 50 60 70 80 90 100 110

Precipitating Protein after unfolding

Nano DSC with a Continuous Capillary Sample CellDSC with a “coin” Shaped Sample Cell

Stable baselineafter unfolding

He

at R

ate

/µJ

s-1

Temperature (˚C)

50

40

30

20

10

0

-10

32

Instrument Control & Data Acquisition SoftwareTheAffinityandNanoinstrumentscontrolanddataacquisitionfunctionsareexecuted

withinaWindows-compatiblesoftware interface, ITCRunorDSCRun. Allexperimental

parameters and sample information are easily entered into an intuitive graphical user

interfaceandcanbesavedasanexperimentaltemplateforfutureuse.

Real-time monitoring of the raw data as the experiment progresses allows rapid

assessmentofthedataqualityandinstrumentperformanceinindividualtabs.Unique

icon-controlled functions, such as immediate baseline subtraction, are always available

on the display.

ITCRun & DSCRun features: •Automaticconfigurationofuserinterfaceforautomatedornon-automated

instruments

•Individualviewingtabsforreal-timemonitoringofinstrumentperformance

characteristics and raw data acquisition

•Easyexperimentsetup

•Directautosamplerprogrammingandcontrolforautomatedinstruments

•SoftwarepassesallexperimentalparameterstoNanoAnalyze™

ITC & DSC SOFTWARE

33Software

25

20

15

10

5

00

-5

-10

-15

-20

-25

-30

-350 1 2 3

No

rma

lize

d F

it(kJ

/mo

l)C

orre

cte

d H

ea

t Ra

te (

µJ/s

)

Time (s)

Mole Ratio

Model Variable ValueIndependent Kd (M) 3.095E-5 n 0.995 ∆H (kJ/mol) -37.94 ∆S (J/mol K) -40.92

Data Analysis with NanoAnalyze™AllITCandDSCrawdatafilesareeasilyandquicklyanalyzedwithapowerfulITC/DSC

data analysis software, NanoAnalyze. Individual window tabs for each processing step

guide the user through the analysis of Individual raw data files or the batch processing

of multiple files.

NanoAnalyze™ features: •EasyimportofallITCandDSCrawdatafiles

•UserselectablefittingmodelsforITCandDSC

•Easysetupofnewfittingmodels

•Drag&Dropsubtractionofbaselineblankfiles

•Powerfulexperimentdesignandoptimizationtool

•Flexibleoverlaygraphsforquickdatacomparisons

•Generatesthermodynamicprofilebargraph

•Easyexportofalldatatodelimitedtextfiles

•Full-featurededitingtoolsforpreparationofpublication

quality images

All instrument control, data acquisition and data analysis software required for ITC and

DSC data are provided with all Affinity and Nano instruments. All software updates and

feature improvements are available on the TA Instruments website.

34

ITC & DSC SPECIFICATIONS

Affinity ITC and ITC Auto Standard Volume Low Volume

MinimumDetectableHeat 0.05µJ 0.05µJ

MaximumMeasurableHeat 5,000µJ 5,000µJ

LowNoiseLevel 0.0014µWatt 0.0014µWatt

BaselineStability 0.02µWatt/hr 0.02µWatt/hr

Temperature Stability ±0.00005°Cat25°C ±0.00005°Cat25°C

TemperatureControl Activeheating&cooling Activeheating&cooling

OperatingTemperature 2to80°C 2to80°C

SampleCellSize 1.0mL 190µL

InjectionSyringeVolume 250µL 250µL

MinimumInjectionVolume 0.01µL 0.01µL

StirringSpeedRange 0–200rpm 0–200rpm

Recommended Stir Speed 75 rpm 75 rpm

ResponseTime 13Sec 11Sec

CellGeometry FixedCylindrical FixedCylindrical

CellComposition 24KGoldorHastelloy 24KGold

Automation Specifications

Autosampler Tray Temperature

ControlRange Ambientto4°C Ambientto4°C

SampleCapicity 96(96-wellplateformat) 96(96-wellplateformat)

AvailableWash/RinseBufferPorts Five(5)onAutosampler Five(5)onAutosampler

35Specifications

Nano ITC Standard Volume Low Volume

MinimumDetectableHeat 0.05µJ 0.05µJ

MaximumMeasurableHeat 3,000µJ 3,000µJ

LowNoiseLevel 0.0014µWatt 0.0014µWatt

BaselineStability 0.02µWatt/hr 0.02µWatt/hr

Temperature Stability 0.0002°Cat25°C ±0.00002°Cat25°C

TemperatureControl Activeheating&cooling Activeheating&cooling

OperatingTemperature 2to80°C 2to80°C

SampleCellSize 1.0mL 190µL

InjectionSyringeVolume 100µL&250µL 50µL

MinimumInjectionVolume 0.06µL 0.06µL

StirringSpeedRange 150–400rpm 150–400rpm

RecommendedStirSpeed 350rpm 350rpm

ResponseTime 13Sec 11Sec

CellGeometry FixedCylindrical FixedCylindrical

CellComposition 24KGoldorHastelloy 24KGold

Nano DSC and DSC Auto

Short-termNoise 0.015µWatts

BaselineStability ±0.028µWatts

Response Time 7 seconds

OperatingTemperature -10°Cto130°Cor160°C

TemperatureScanRate upto2°C/minute

PressurizationPerturbation Built-inupto6atmospheres

CellVolume 300mL

CellGeometry Fixedcapillary

CellComposition Platinum

HeatMeasurementType PowerCompensation

AutomationSampleCapacity 2standardplatesx96wellsx1000μL/well

SampleTrayTemperatureControlRange 4°CtoAmbient

AvailableWash/RinseBufferPorts 4forSample/ReferenceCells;2forSampleHandlingSyringe

36

References

Garbett,N.,DeLeeuw,L.andJ.B.Chaires.MCAPN-2010-04. High-throughputDSC:AComparisonoftheTAInstrumentsNanoDSCAutosamplerSystem™withtheGEHealthcareVP-CapillaryDSC™(2010)

Demarse,N.andL.D.Hansen.MCAPN-2010-01. AnalysisofBindingOrganicCompoundstoNanoparticlesbyIsothermalTitrationCalorimetry(ITC).(2010)

Quinn,C.F.MCAPN-2010-02.AnalyzingITCDatafortheEnthalpyofBindingMetalIonstoLigands.(2010)

Quinn,C.F.andL.D.Hansen.MCAPN-2010-03.PressurePerturbationCalorimetry:DataCollectionandFitting.(2010)

Román–Guerrero,A.,Vernon–Carter,E.J.andN.A.Demarse.MCAPN-2010-05.ThermodynamicsofMicelleFormation.(2010)

TAInstruments,MicrocalorimetryTechnicalNoteMCTN-2010-02. AdvantagesofUsingaNanoDSCwhenStudyingProteinsthatAggregateandPrecipitatewhenDenatured.(2010)

TAInstruments,MicrocalorimetryTechnicalNote,MCTN-2010-03.HowtoChooseanITCCellVolume.(2010)

Baldoni,D.,Steinhuber,A.,Zimmerli,W.andA.Trampuz.AntimicrobAgentsChemother54(1):157-63.InvitroactivityofgalliummaltolateagainstStaphylococciinlogarithmic,stationary,andbiofilmgrowthphases:comparisonofconventionalandcalorimetricsusceptibilitytestingmethods.(2010)

Choma,C.T.MCAPN-2010-01.CharacterizingVirusStructureandBindingbyCalorimetry.(2009)

Wadsö,L.andF.G.Galindo.FoodControl20:956–961.Isothermalcalorimetryforbiologicalapplicationsinfoodscienceandtechnology.(2009)

Baldoni,D.,Hermann,H.,Frei,R.,Trampuz,A.andA.Steinhuber.JClinMicrobiol.7(3):774-6. PerformanceofmicrocalorimetryforearlydetectionofmethicillinresistanceinclinicalisolatesofStaphylococcusaureus.(2009)

Trampuz,A.,Piper,K.E.,Hanssen,A.D.,Osmon,D.R.,Cockerill,F.R.,Steckelberg,J.M.andR.Patel.JClinMicrobiol.44(2):628-631. Sonicationofexplantedprostheticcomponentsinbagsfordiagnosisofprostheticjointinfectionisassociatedwithriskofcontamination.(2006)

Microcalorimetry-ANovelMethodforDetectionofMicroorganismsinPlateletConcentratesandBloodCultures.AndrejTrampuz,SimoneSalz-mann,JeanneAntheaume,RenoFrei,A.U.DanielsUniversityofBasel&UniversityHospitalBasel,Switzerland(2006)

DataprovidedbySvensson,BodycoteMaterialsAB,Sweden(2003)

WingborgandEldsater,Propellants,ExplosivesandPyrotechnics,27,314-319,(2002).

Schmitt,E.A.,Peck,K.,Sun,Y.andJ-MGeoffroy.ThermochimicaActa

380(2):175-184.Rapid,practicalandpredictiveexcipientcompatibilityscreeningusingisothermalmicrocalorimetry.(2001)

Schmitt,Peck,Sun&Geoffroy,Thermochim.Acta,380,175-183,(2001).

ThermometricApplicationNote22034(2001).

Hogan,S.E.&G.Buckton.Int.J.Pharm.,207,57-64.Thequantificationofsmalldegreesofdisorderinlactoseusingsolutioncalorimetry.(2000)

Chemical&EngineeringNews,June18,2007,Page31.Hongisto,Lehto&Laine,Thermochim.Acta,276,229-242,(1996).

Bermudez,J.,Bäckman,P.andA.Schön.Cell.Biophys.20,111-123. MicrocalorimetricEvaluationoftheEffectsofMethotrexateand6-ThioguanineonSensitiveT-IymphomaCellsandonaMethotrexate-ResistantSub-line.(1992)

Bystrom,ThermometricApplicationNote22004,(1990).

ThermometricAppl.Note22024.

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