In The Name of AllahIn The Name of Allah

Thermodynamics is an impressive term that might seem more than just a little intimidating at first.

Luckily, like many things, once you get to know it a bit, it’s not as mysterious and difficult as it seemed at first. It’s really all about energyIt’s really all about energy.

The word "thermodynamics" comes from Greek roots meaning heat (thermothermo) and energy, or power (dynamicsdynamics).

So, Thermodynamics is really just the study of heat and energyThermodynamics is really just the study of heat and energy, and how it relates to the matter in our universe.

Thermodynamics is used in many fields of study, such as physics and physics and engineeringengineering, to understand physical processesunderstand physical processes.

Not surprisingly, it is also used by biochemists to understand processes and by biochemists to understand processes and chemical reactionschemical reactions that occur in living organisms.

Despite all these applications, the basic tenets of thermodynamics can be stated in just a few laws.

There are three laws of thermodynamicsthree laws of thermodynamics, the first two of which are of most interest to biochemists.

This is the law of the conservation of energythe law of the conservation of energy. It states that energy can neither be created, nor can it be destroyed.

This means that the total amount of energy in the universe always remains conserved, or constant. However, energy energy can be changed from one form to anothercan be changed from one form to another.

There are many different forms of energy, some of which may be more useful than others for a particular process.

ElectricalElectrical, chemicalchemical, and mechanicalmechanical are all examples of different forms of energy.

The definition of energy is the ability to do workthe ability to do work.

This is the law of increasing entropythe law of increasing entropy.

It states that the entropy of the universe increases with the entropy of the universe increases with every physical process every physical process (change) that occurs.

Entropy refers to the level of disorder, randomness, or chaos, of a system.

The higher the randomnessthe randomness of a system, the higher its entropyentropy.

The more organized a system, the lower its entropy.

A "system""system" is the part of the world we are interested in.

It can be very small, like a single molecule, or as large as the entire universe.

A drop of dye placed in a cup of water will eventually result in an evenly colored solution, even if we never stir the liquid.

The dye molecules distribute as evenly as possible throughout the volume of water.

An example of this is the conversion of the the conversion of the energy in gasoline to energy in gasoline to powerpower an automobile.

Only about 20% of the energy results in motion of the vehicle, while the rest of the energy is lost as heatlost as heat.

This idea that entropy is always increasing can be a bit confusinga bit confusing. The second law states that the entropy of the universe must increase with each the universe must increase with each processprocess. However, this is not the same as saying the entropy of a system must increase the entropy of a system must increase with each processwith each process. The system can become more orderedmore ordered, but the price is that the surroundings must become much more disorderedmore disordered, so that there is still an overall increase in entropy for the universe.

In the previous section, we discussed that spontaneous reactions always proceed in a direction that will give the a direction that will give the products less potential products less potential energyenergy, or energy available to do work, than they started with. This means that in a a spontaneous chemical spontaneous chemical reaction, energy is releasedreaction, energy is released, and the products of the the products of the reaction have less energy reaction have less energy than the original reactantsthan the original reactants.

Let’s look at a hypothetical spontaneousspontaneous chemical reaction:

The quantity of usable energy (chemical potential) in a reaction is called the Gibbs Free EnergyGibbs Free Energy (ΔG). ΔG is the difference between the energy the difference between the energy contained in the products of a reaction and the reactantscontained in the products of a reaction and the reactants:

ΔG = (energy of products) - (energy of reactants)

Chemical reactions are classified as being either "exergonic""exergonic" or "endergonic""endergonic". That just means that a reaction can either release energyrelease energy useful for work (an exergonic reaction) or requires energyrequires energy to proceed (an endergonic reaction).

The spontaneous reaction above is an exergonic reactionspontaneous reaction above is an exergonic reaction. Note how for an exergonic reaction, ΔG will be negative. Thus, a negative ΔG value tells you that that reaction is possible.

Exergonic Reaction Endergonic Reaction

Spontaneous Not Spontaneous

Release Free Energy ( -ΔG) Consume Gibbs Free Energy (+ΔG)

Products have less energy than reactants

Products have more energy than reactants

For example, here is the reaction when the high energy

compound ATP is hydrolyzed to release an inorganic

phosphate molecule and energy: ATP + H2O ADP + Pi         ΔG

= -30.5 kJ/mol Note that when the reaction releases energy,

ΔG is negative! When the reaction is written in reverse, the

sign of DG changes: ADP + Pi → ATP + H2O         ΔG = +30.5

kJ/mol

For example, here is the reaction when the high energy compound ATP is hydrolyzed to release an inorganic phosphate molecule and energy:

ATP + H2O → ADP + Pi ΔG = -30.5 kJ/mol

Note that when the reaction releases energy, ΔG is negative!

When the reaction is written in reverse, the sign of ΔG changes:

ADP + Pi → ATP + H2O ΔG = +30.5 kJ/mol

All chemical reactions reach a point where they settle they settle downdown and won’t go any further.

At that point, the reaction is said to be at equilibrium.

This point is not when all the reactants have been used up (this is actually rarely seen), nor when all the components of the reaction are present in equal quantities.

How much product and how much reactant are present at equilibrium depends on the specific chemical depends on the specific chemical properties of each of the compoundsproperties of each of the compounds involved.

The equilibrium constant, KKeqeq, is a measure of the measure of the concentrations of the reactants and productsconcentrations of the reactants and products when the chemical reaction has reached equilibrium.

The equilibrium constant (Keq) is defined by the ratio of products to reactants when the reaction is at equilibrium.

That is, Keq is calculated from the concentrations of the reactants and products when the reaction has finishedthe reaction has finished (reached equilibrium).

You can do the reaction many times, starting out with different concentrations of reactants and products each time, but at the end of the reaction, the ratio of products to the ratio of products to reactants will always end up the samereactants will always end up the same.

Let’s look at our hypothetical reaction from the discussion on Keq again:

If the reaction above proceeds so that at equilibrium, the concentration of products the concentration of products is greater than the concentration of reactantsis greater than the concentration of reactants, then the reaction is said to lie "to the "to the right"right".

In that case, the concentrations of C and D will be higher than the concentrations of reactants A and B, and the Keq will be greater than 1greater than 1.

Remember from our look at the Gibbs Free Energy, that if the formation of products is favored, then the ΔG is negative.

On the other hand, if the reaction is at equilibrium when there are still more reactants left over than products (lies "to the left""to the left"), then the concentrations of A and B will always be larger than the concentrations of C and D, and the Keq will be less less than 1than 1.

Again, from our look at Gibbs Free Energy, when the formation of reactants is energetically favored, the ΔG is positive.

Thus, there is a relationship between Keq and relationship between Keq and ΔΔGG.

Direction of Reaction Keq ΔG

Toward forming more products >1 negative (-)

Toward forming more reactants <1 positive (+)

Products and reactants equal 1

There is an equation that relates Gibbs Free

Energy to the Equilibrium Constant: ΔG = -2.3RT

log Keq R is a constant value (the gas constant,

8.3 J/mol/K )T is the temperature at which the

reaction occurs (in Kelvin)

There is an equation that relates Gibbs Free Energy to the Equilibrium Constant:

ΔG = -2.3RT log Keq

R is a constant value (the gas constant, 8.3 J/mol/K )T is the temperature at which the reaction occurs (in Kelvin)

Why is all this important? The idea that there is a relationship between the concentrations of the reactants and products and the direction in which a reaction proceeds has important consequences for chemical reactions. Many metabolic reactions in our bodies (reactions that produce energy, or create building blocks to build up our bodies) can go forwardforward or backwardbackward, depending on the surplus of reactants, or the demand for their productsthe demand for their products.

Let’s say there is a small store that sells products on one shore of a lake. Because it’s small, the store owner keeps extra inventory in a warehouse on the other side of the lake. If the warehouse is full but the store is empty, the boat moves from the warehouse to the store, so that there is more product to sell. This is similar to a biochemical reaction proceeding in an environment where the the reactant-to-product ratio is reactant-to-product ratio is greater than it is at greater than it is at equilibriumequilibrium. In other words, there is too much reactant (stuff at the warehouse), and not enough product (at the store). The reaction (boat) spontaneously proceeds toward equilibrium, which in this case is "to the "to the right"right".

On the other hand, if it’s right after Christmas and suddenly there are a lot of returns, there is too much product at the store. The owner has to send some back to the warehouse. This is similar to a situation where a biochemical reaction is proceeding in an environment where the reactant-to-product the reactant-to-product ratio is smaller than at ratio is smaller than at equilibriumequilibrium. In other words, there is too much product while the warehouse sits empty. Again, the reaction moves spontaneously towards equilibrium, but in this case, that means the boat goes in the opposite direction, "to "to the left"the left".

One last thing to remember; the word "spontaneous""spontaneous" sure makes it sound like these reactions happen fastthese reactions happen fast.

But the truth is, even when a reaction is thermodynamically possiblethermodynamically possible, (has a negative ΔG value), it often happens very slowlyit often happens very slowly. This is especially true of many biochemical reactions that occur under physiological conditions, that is, within our bodies.

Think of what would happen to our store owner if his boat were to break down.

He might try to float his packages across the lake, but it could take forever for them to get to the other side.

So, even if the warehouse is full, the store will get little or no product unless the boat moves.

Enzymes act in much the same way in biochemical reactionsEnzymes act in much the same way in biochemical reactions.

Metabolic reactions in our bodies are catalyzed, or helped along, by special proteins called enzymes.

They greatly speed up the rate at which reactions occur, so that reactions that They greatly speed up the rate at which reactions occur, so that reactions that are thermodynamically possible but very slow can now proceed at a rate that are thermodynamically possible but very slow can now proceed at a rate that makes them useful for sustaining life.makes them useful for sustaining life.

Even though the universe is continually becoming more disordered (following the second law of thermodynamics), not everything is utter chaos.

For instance, we know that our bodies are highly organized. Each of our different organs and tissues performs unique functions, and even at the cellular level molecules are partitioned into organelles and compartments.

Because they possess such highly organized structure, living organisms are said to have a relatively low amount of entropy.

To sustain life, organisms need to build up a complex body from more basic building blocks. Complex proteins are made from long chains of amino acids, precisely and intricately folded.

DNA is similarly comprised of long chains of nucleic acids.

We know from the second law of thermodynamics that the reactions that create these molecules of life are certainly not spontaneousnot spontaneous; energy input is required to make them go.

Organisms do not just magically assemble themselves. But how do living things deliver the needed energy to such body-building chemical reactions?

We have already discussed that some reactions are exergonic (they release energy that might be useful for work), while others are endergonic (they need energy to make them go).

To get the energy to those endergonic reactions, they are paired up with energy-releasing exergonic reactions.

Like a locomotive that gets the train car over the hill, an exergonic reaction can "pull" an endergonic reaction along to its destination (products).

The reactions can be hooked together, or coupled, via a common intermediate.

Thermodynamics lets us predict that the reaction will proceed if the overall ΔG of the two reactions is negative. When two reactions are coupled, the overall ΔG is the sum of the DGs of the component reactions.

For example, in the biochemical pathway that breaks down glucose for energy, two enzymes work one after the other to create a high-energy ATP molecule:

Enzyme 1+ cofactor: (1) Glyceraldehyde-3-phosphate + Pi ↔ 1,3 bisphosphoglycerate (ΔG = 0

kJ/mol)

Enzyme 2 (2) 1,3 bisphosphoglycerate + ADP ↔ 3-phosphoglycerate + ATP (ΔG = -16.7

kJ/mol)

The first and second laws of thermodynamics are useful in helping us to understand bioenergetics, the flow of energy through living systemsthe flow of energy through living systems.

They can help us determine whether a physical process, such as a biochemical reaction, is possible. Reactions that require energy input will not proceed Reactions that require energy input will not proceed naturally, or spontaneouslynaturally, or spontaneously.

However, such energy-requiring, endergonic reactions can be coupled to spontaneous, exergonic reactions.

This linking together of two chemical processes allows the energy-requiring reactions to proceed by "borrowing" energy from energy-releasing reactions.

We can calculate whether a reaction is spontaneous or not with the Gibbs Free Energy, ΔG.

The ΔG value and the equilibrium constant, Keq, allow us to predict whether the products or the reactants will be more abundant at equilibrium.

Understanding Keq and Le Chatelier’s Principle also allows us to predict which which way the reaction will go to reach equilibrium when extra reactants or products way the reaction will go to reach equilibrium when extra reactants or products are presentare present.

However, Thermodynamics is not useful for determining reaction ratesThermodynamics is not useful for determining reaction rates.

Refernce:

http://www.wiley.com/college/boyer/0470003790/reviews/reviews.htm

CalorimetryCalorimetry is a primary technique for measuring the thermal properties of materialsfor measuring the thermal properties of materials to establish a connection a connection

between temperature and specific physical propertiesbetween temperature and specific physical properties of substances and is the only method for direct the only method for direct

determination of the enthalpydetermination of the enthalpy associated with the process of interest.

CalorimetersCalorimeters are frequently used in "Chemistry" , "Biochemistry", "Cell Biology", "Biotechnology", "Pharmacology"

, and recently in "Nanoscience" to measure thermodynamic properties of the biomolecules and nano-sized

materials.

Amongst various types of calorimeters, differential scanning calorimeter (DSC)differential scanning calorimeter (DSC) is a popular one, which is a thermal

analysis apparatus measuring how physical properties of a sample change along with temperature against time.

In other words, the device is a thermal analysis instrument that determines the temperature and heat flow the device is a thermal analysis instrument that determines the temperature and heat flow

associated with material transitionsassociated with material transitions as a function of time and temperature.

During a change in temperature, DSC measures a heat quantity which is excessively radiated or absorbed by the DSC measures a heat quantity which is excessively radiated or absorbed by the

samplesample; on the basis of a temperature difference between the sample and the reference material.

Based on mechanism of operation, differential scanning calorimeters can be classified into two types: 1)1) Heat flux

DSCs, and 2)2) Power compensated DSCs.

In a heat flux DSCheat flux DSC, the sample material, enclosed in a pan and an empty reference pan are placed on a thermoelectric disk on a thermoelectric disk surrounded by a furnacesurrounded by a furnace.

The furnace is heated at a linear heating rate and the heat is transferred to the sample and reference pan through through thermoelectric diskthermoelectric disk.

However, owing to the heat capacity of the sample there would be a temperature difference between the sample and reference pans, which is measured by area thermocouplesby area thermocouples and the consequent heat flow is determined by the thermal equivalent of by the thermal equivalent of Ohm’s lawOhm’s law:

Where qq is "sample heat flow", ΔΔTT is "temperature difference between sample and reference” and RR is "resistance of thermoelectric disk".

Differential scanning calorimetry (DSC) is a thermodynamical tool for a thermodynamical tool for direct assessment of the heat energy uptake which occurs in a sample direct assessment of the heat energy uptake which occurs in a sample within regulated increase or decrease in temperaturewithin regulated increase or decrease in temperature. The calorimetry is particularly applied to monitor the changes of phase transitionsto monitor the changes of phase transitions.

DSC is commonly employed for study of biochemical reactions which is for study of biochemical reactions which is named as a single-molecular transition of a molecule from one named as a single-molecular transition of a molecule from one conformation to anotherconformation to another. Thermal transition temperaturesThermal transition temperatures "(melting points)" of the samples are also determined in solution, solid or mixed phases like suspensions.

In a basic DSC experiment, energy is introduced simultaneously into a energy is introduced simultaneously into a sample cellsample cell (which contains a solution with the molecule of interest) and a a reference cell reference cell (containing only the solvent).

Temperatures of both cells are raised identically over timeTemperatures of both cells are raised identically over time. The differenceThe difference in the input energy required to match the temperature of the sample to that of the reference, would be the amount of excess heat either absorbed the amount of excess heat either absorbed or released by the molecule in the sampleor released by the molecule in the sample (during an endothermic or exothermic process respectively).

Due to the presence of molecule of interestDue to the presence of molecule of interest, more energymore energy is required to bring the sample to the same temperature as the reference; hence the the concept of heat excess comes into the pictureconcept of heat excess comes into the picture (Fig. 1).

Experimental setup for a Experimental setup for a differential scanning differential scanning calorimetry experimentcalorimetry experiment.

The amount of heat required to increase the temperature by the same increment (ΔTΔT) of a sample cell (qsqs) is higher than that required for the reference cell (qrqr) by the excess heat absorbed by the molecules in the sample (ΔqΔq).

The resulting DSC scans with the reference subtractedsubtracted from the sample shows how this excess heat changes as a function of temperature.

DSC is capable of elucidating the factors that contribute to the folding and the factors that contribute to the folding and

stability of biomoleculesstability of biomolecules.

Changes in the heat capacityChanges in the heat capacity are believed to originate from the disruption originate from the disruption

of the forces stabilizing native protein structureof the forces stabilizing native protein structure.

For example this includes:

› "van der Waals", "hydrophobic and electrostatic interactions",

"hydrogen bonds", "hydration of the exposed residues conformational

entropy", "the physical environment (such as pH, buffer, ionic

strength, excipients)".

Therefore, thermodynamic parameters obtained from DSC experimentsthermodynamic parameters obtained from DSC experiments,

are quite sensitive to the structural state of biomoleculequite sensitive to the structural state of biomolecule.

› Any change in the conformation, would affect the position, sharpness and the shape of Any change in the conformation, would affect the position, sharpness and the shape of

transition(s) in DSC scans.transition(s) in DSC scans.

In a DSC experiment, thermodynamic parameters are associated with thermodynamic parameters are associated with

heat-induced macromolecular transitionsheat-induced macromolecular transitions.

For a typical macromolecule, the molar heat capacity is measured as a the molar heat capacity is measured as a

function of temperaturefunction of temperature; subsequently yielding the following

thermodynamic parameters:

› The partial heat capacity of a molecule

› Change in enthalpy (ΔH) of the transition

› Change in entropy (ΔS) of the transition

› Change in heat capacity (ΔCp) of the transition

› Melting point (Tm) of the transition

› Absolute heat capacity

Gill et al., Differential Scanning Calorimetry Techniques:

Applications in Biology and Nanoscience. Journal of Biomolecular

Techniques 2010.

Donald et al., Calorimeters for Biotechnology. Thermochimica

Acta 2006.

Thanks for Your AttentionsThanks for Your Attentions

In a DSC experiment, thermodynamic

parameters are associated with heat-heat-

induced macromolecular transitionsinduced macromolecular transitions.

For a typical macromolecule, the molar the molar

heat capacityheat capacity is measured as a function of

temperature; subsequently yielding the

following thermodynamic parameters.

The heat capacity of the solution containing a macromolecule is

measured with respect to the heat capacity of buffer in the the heat capacity of buffer in the

absence of macromoleculesabsence of macromolecules.

Hence the instrument measures only part of what could be only part of what could be

actually measuredactually measured which is the difference between sample and

reference cells.

The sample could be a protein, tRNA, a protein–DNA complex, a

protein–lipid complex, or something else.

The heat capacity at constant pressure is a temperature a temperature

derivative of the enthalpy functionderivative of the enthalpy function (Cp = (H/T)p and thus the

enthalpy function can be measured through integration of the

heat capacity (H(T)= Cp(T)dT + H(T0)).

The partial molar heat capacity functions of (a) barnase (Mw=12.4kDa) and (b) ubiquitin (Mw=8.4 kDa), in solutions with different pH. The dashed lines represent the partial molar heat capacity of native and unfolded proteins. Data are adapted from "Privalov PL, Dragan AI. Microcalorimetry of biological macromolecules. Biophys Chem. 2007; 126: 16–24".

For any biomolecule in aqueous solution, there would be equilibrium equilibrium

between the native conformationbetween the native conformation (folded) and its denatured stateand its denatured state

(unfolded).

Stability of the native conformation is based on the extent of Gibbs free the extent of Gibbs free

energy (ΔG) of the systemenergy (ΔG) of the system, and thermodynamic relationships between

changes in the enthalpy (ΔH) and entropy (ΔS).

A positive magnitude of ΔG represents higher stabilitypositive magnitude of ΔG represents higher stability of the native

conformation than that of the denatured state.

During the unfolding process of a protein, forcesforces that play key role in

stabilization need to be broken.

At temperatures where entropy is the dominant factorwhere entropy is the dominant factor, conformational

entropy overcomes the stabilizing forces, leading to unfolding of protein.

Differential scanning calorimetry measures ΔH of unfolding due to ΔH of unfolding due to

heat denaturationheat denaturation.

The transition midpoint Tm is considered as the temperature where the temperature where

50% of the protein owns its native conformation and the rest 50% 50% of the protein owns its native conformation and the rest 50%

remains denaturedremains denatured.

Higher TmHigher Tm values would be representative of more stablemore stable molecule.

During the same experiment, DSC is also capable of measuring the the

change in heat capacitychange in heat capacity (ΔCp).

Associated with protein unfolding process, heat capacity changes

occur as a result of changes in hydration of side chainschanges in hydration of side chains which are

buried in the native conformation, but become exposed to the

solvent in denatured state.

Calorimetric enthalpy (∆Hcal) means the total the total

integrated zone below the thermogram peakintegrated zone below the thermogram peak which

indicates total heat energy uptake by the sampletotal heat energy uptake by the sample after

suitable baseline correction affecting the transition.

Van’t Hoff enthalpy (∆HVH) is an independent an independent

measurement of the transitional enthalpymeasurement of the transitional enthalpy according to

the model of the experiment. ∆HVH is determined

through the shape analysis of an experimental graph

of versus T.

The state of the transition is evaluated by comparing of ∆HVH and

∆Hcal.

If ∆HVH is equal toequal to ∆Hcal, the transition occurs in a two-state mode.

› In such process, manful thermodynamical results are determined through

van’t Hoff measurements of equilibrium results.

When ∆HVH is more thanmore than ∆Hcal, the intermolecular cooperation is

shown which is exposed for example as aggregation.

› Comparison between ∆HVH and ∆Hcal also indicates the cooperative the cooperative

nature of the transitionnature of the transition. Particularly, ∆HVH/ ∆Hcal ratio gives an estimation

from the fraction of the structure which is melted as a thermodynamical

value. The value is also named as the size of the cooperative unitthe size of the cooperative unit.

Heat capacity change (∆Cp) for transitional state is obtained through the

difference between pretransitional and posttransitional baselinesdifference between pretransitional and posttransitional baselines of a DSC process.

The curve of CpCp against TT can be changed to versus T through dividing the raw

Cp value by T and drawing the results as a function of T.

By integration, this curve results the transition entropy (∆S)the transition entropy (∆S) which is expressed as

(∆S) = ∫( ) dT.

› Hence an individual DSC thermogram can result ∆H, ∆S, and ∆CpHence an individual DSC thermogram can result ∆H, ∆S, and ∆Cp.

After knowing the above data, transition free energy (∆G) can be given at each

temperature (T) through the famous thermodynamical equation: ∆G = ∆H - T∆S∆G = ∆H - T∆S

Although ∆S and ∆G can be obtained by DSC results, the values are more

unreliable than the ∆H and ∆Cp values determined directly because of coupling because of coupling

and propagating of errorsand propagating of errors.

Apparent heat capacityApparent heat capacity can be obtained by DSC results.

› It includes the contribution of water displacement by the protein in the contribution of water displacement by the protein in

sample cellsample cell which could have even negative value.

Correction for the water displacement effect and normalization to a mole

of protein offers the absolute heat capacityabsolute heat capacity.

The value is obtained from doing a series of DSC measurements at from doing a series of DSC measurements at

different protein concentrationsdifferent protein concentrations.

Determined absolute heat capacities by DSC could be employed to to

characterize long-range interactions and cooperative phenomenacharacterize long-range interactions and cooperative phenomena which

have been shown to occur in denatured proteins.

Analysis of Proteins

Differential Scanning Calorimetry of Nucleic Acids

Analysis of Lipids

Analysis of Carbohydrates

Analysis of Monoclonal Antibodies

Gill et al. Differential Scanning Calorimetry:

Application in Biology and Nanoscience. J.

Biomol. Tech., 2010.

Privalov PL, Dragan AI. Microcalorimetry of

Biological Macromolecules. Biophys Chem., 2007.

"Thanks for Your Attentions""Thanks for Your Attentions"