Differential scanning calorimetry
Measure the heat absorbed as a protein unfolds.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
1Measuring unfolding as heat
A differential scanning calorimeter heats two matched cells at the same constant rate: one holds the protein in buffer, the other the same buffer alone. Whatever extra power the sample cell needs to keep pace is the heat the protein absorbs, and dividing it by the scan rate gives a heat capacity.
2The calorimetric criterion
Comparing the two enthalpies is the test of whether unfolding is two-state. If one cooperative unit unfolds all at once, the shape and the area must agree and ΔHvH/ΔHcal is close to 1.
Tm
Where the excess heat capacity is maximal, close to the midpoint of unfolding.
ΔHcal
Model-free: heat absorbed per mole, so it depends directly on the concentration.
ΔHvH
From the sharpness of the peak under a two-state model; independent of concentration.
ΔCp
The rise from native to unfolded baseline; the least precise of the four.
3Reversibility and aggregation
Equilibrium thermodynamics applies only if the protein refolds when cooled. Many proteins do not: once unfolded they aggregate, and aggregation releases heat, so the trace dips after the peak, the area shrinks, and Tm becomes a property of the scan rate rather than the protein.
4Where the baseline comes from
The protein's signal is a few tens to a few hundred µcal/K on top of an instrument baseline of similar size, so the baseline decides the answer. The reference cell must contain the protein's own final dialysate, since any difference in salt, glycerol or pH between the cells adds heat capacity that no subtraction removes.
Key terms
Practise these as flashcards →The heat capacity of the protein above its native baseline, in kJ/mol/K, after subtracting the buffer scan and dividing by moles of protein.
The area of the transition peak: heat absorbed per mole of protein, independent of any model.
The enthalpy implied by the sharpness of the transition under a two-state model.
The calorimetric criterion: about 1 for two-state unfolding, below 1 for multi-domain or intermediate-containing transitions, above 1 for coupled association or a concentration error.
The heat-capacity change on unfolding, mostly from hydration of exposed nonpolar groups; it curves the stability against temperature.
A scan with buffer in both cells, run until reproducible, that defines the instrument baseline to subtract.
A second heating of the same sample after cooling in the cell; the standard test of reversibility.
Reversible unfolding followed by an irreversible step (often aggregation), used to analyse scan-rate-dependent DSC transitions.
When to use it
- Obtaining model-free thermodynamic parameters of unfolding (ΔH, ΔCp and Tm) for a purified protein, for example to compare mutants or explain a stability difference.
- Showing whether a protein unfolds as one cooperative unit or as several domains, from the ratio of van 't Hoff to calorimetric enthalpy and from deconvolution of overlapping peaks.
- Formulation and developability work on biologics such as antibodies, where several domain transitions and their dependence on buffer and excipients need to be resolved.
- Measuring ligand-induced stabilisation in solution where optical signals are unavailable or unreliable, for instance with coloured or turbid samples.
Compared with alternatives
Far higher throughput and much less protein for Tm and ligand shifts, but no enthalpy or ΔCp and dependent on a dye or tryptophan signal; prefer it for screening.
Follows unfolding through a structural signal with less protein and tells you what changes, but gives only a van 't Hoff enthalpy.
The calorimetry of binding at constant temperature: KD, stoichiometry and binding enthalpy rather than unfolding.
Chemical denaturation with fluorescence gives ΔG at 25 °C directly; combined with DSC's ΔH and ΔCp it gives a full stability curve.