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•09/11/2010
•1
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,
•09/11/2010
•2
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
0
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12000
14000
16000
18000
0 1 2 3 4
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
0
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0 1 2 3 40
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0 1 2 3 4
Ener
gy (J
/g)
Time (d)
•09/11/2010
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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)
0
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0 5000 10000 15000 200000
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0 5000 10000 15000 20000
Hea
t flo
w (µ
W/g
)
Energy (J/g)
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
0
1
2
3
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
•09/11/2010
•8
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
•09/11/2010
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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)
•09/11/2010
•10
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
•09/11/2010
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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
•09/11/2010
•12
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)
•09/11/2010
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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
-1
0
1
2
3
4
5
6
7
0 1000 2000 3000 4000 5000 6000Time / s
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
�
Continuous titrationBaCl2 titrated into 18-crown-6
0
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25
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40
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50
200 300 400 500 600 700 800 900 1000Time (seconds)
Hea
t Rat
e (µ
W)
Raw DataFit
n = 1.01Ka = 5.97 x 103 M-1
∆H = -31.4 kJ mol-1
2'-CMP titrated into RNase A
0
1
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10
200 300 400 500 600 700 800 900 1000Time (seconds)
Hea
t Rat
e (µ
W)
Raw DataFit
n = 1.04Ka = 1.12 x 106 M-1
∆H = -73.4 kJ mol-1
•09/11/2010
•17
• 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.
�
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
•09/11/2010
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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
0
2
4
6
8
10
12
14
Cp
(kca
l/mol
e/o C
)
Temperature ( oC)
∆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.
-10
0
10
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30
40
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60
70
80
35 45 55 65 75 85Temperature / °C
Exce
ss C
p / k
J m
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
•09/11/2010
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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
•09/11/2010
•23
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.
Qualche domanda?
Grazie per l’attenzione