Differential Scanning Calorimetry (DSC) is the only analytical method that directly measures the enthalpy change (∆H) of a thermal transition, such as the thermal denaturation of a protein, nucleic acid, or other biopolymer.

What is ∆H?

∆H is the total energy taken up in raising the system to temperature T at constant pressure. For a protein, this means the energy (heat) spent to unfold it, and the ∆H is positive, representing an endothermic process. This energy is associated with all the atomic and molecular motions as well as the energy of the bonds keeping the protein in folded conformation. 

∆H is calculated by integrating the area under the thermogram (see figure below), and is represented in calories (or joules) per mole of protein. As the protein is exposed to increasing temperatures during a DSC experiment, the protein begins to thermally denature, as the non-covalent bonds are broken. The ∆H is related to the number of bonds that are needed to keep the protein in its native (folded) conformation.

∆H depends on how accurately we measure the total protein concentration. If protein concentration is not accurately determined, the calculated ∆H value will be impacted.

What does the ∆H value tell us in practice?

When you compare the DSC results of different proteins, the protein with the greater ∆H value is not necessarily more stable than protein with the small ∆H. Since ∆H is normalized per total molar protein concentration, the value will often be proportional to the size of the protein. Most proteins have the same density of bonds (bonds per volume). It is reasonable to expect that a protein with a larger molecular weight will also have a larger ∆H.

∆H is dependent on the percentage of native protein in solution

An important consideration is that DSC only measures the ∆H value for protein that is initially in its folded (native) conformation. The magnitude of ∆H depends on the concentration of the folded fraction. If the initial folded protein fraction is less than 100% of the total protein concentration, the calculated ∆H value will be correspondingly smaller.

The figure below shows DSC thermograms for the same protein, measured at different times during storage. The blue thermogram is for freshly-prepared protein which is 100% native (folded) protein. As the protein sample deteriorates during storage, the fraction of native protein in the solution begins to decrease, resulting in a decrease of enthalpy in the DSC thermograms. We can use the relative ∆H values from the different themograms to estimate the folded protein fraction for each sample, when we have a reference DSC thermogram with 100% native protein.

In this example, the ∆H for the sample with the green thermogram has 50% the ∆H of the blue sample, so it is 50% folded protein. The orange sample has 25% folded protein, and the red sample has 10% folded protein, relative to the blue thermogram.

DSC thermograms of protein with different fractions of a folded protein. The ∆H values (from the integrated areas under the curves) for each thermogram are: blue – 100 kcal/mole; green – 50 kcal/mole; orange – 25 kcal/mole; red – 10 kcal/mole.

Calorimetric and van’t Hoff enthalpies

So far in this blog, we have written about the “calorimetric” enthalpy that is directly measured by DSC, and is often represented as ∆Hcal. There is another type of enthalpy we can calculate from DSC data, the van’t Hoff enthalpy –  HvH.  This value is available from the DSC Non-Two-State model fit. ∆HvH is also the enthalpy that is determined from any non-calorimetric (indirect) thermal melting technique, such as circular dichroism.

With DSC , ∆Hcal is determined only by the area under a transition peak, while ∆HvH is determined only by the shape of the transition peak. The sharper the transition, the larger ∆HvH is, and vice versa. ∆Hcal is concentration dependent, but HvH is not. 

Usually, a ∆Hcal /∆HvH ratio equal to 1 is taken as an indication that the transition under study conforms to the two-state unfolding mechanism. A ∆Hcal /∆HvH ratio greater than one as an indication of the presence of significantly populated intermediates; and a ∆Hcal /∆HvH ratio smaller than one as an indication of intermolecular interactions.

Using the ∆Hcal /HvH we can estimate that a large fraction of the protein is inactive. If we have a simple single-domain protein, and we assume no intermediates, it may be expected that its unfolding will have a ratio of ∆Hcal /HvH  not too far from 1. So, a  ∆Hcal significantly lower than the ∆HvH , may indicate that a large fraction of the protein is already inactive. 

To summarize, DSC analysis of the ∆H data can provide insights into the unfolding mechanism of proteins, and how much protein is in its native conformation.

Further reading