Equipment available: Malvern VP Capillary DSC
Equipment location: New Biochemistry 00-059
Equipment coordinator: Dr David Staunton
Equipment charge: £25 per sample
Sample requirements: 0.4ml of approx. 0.01M and 4ml matching buffer
Differential Scanning Calorimetry
Calorimetry is the measurement of heat evolved or absorbed during a chemical or physical change in a sample. Differential Scanning Calorimetry (DSC) measures heat changes that occur during a controlled increase or decrease in temperature and determines the heat capacity of a molecule, in aqueous solution, as a function of temperature. It can be used to measure directly the stability and unfolding of proteins, lipid membranes or nucleic acids. DSC determines the heat capacity of a molecule, in aqueous solution, as a function of temperature but as a standard protein concentration used would be 20 µM compared to the water concentration of 55M the signal from the buffer alone must be subtracted from the sample in order to see any signal from the biological molecules. To achieve this two cells are used, one containing the buffer only (reference) and the other the buffer and biological molecule (sample). Subtraction of the heat capacity of the two samples gives the heat capacity contribution of the protein alone. The DSC cells are contained in a thermal insulation jacket (adiabatic) chamber. Heaters are used to transfer heat energy to the cells (to raise the temperature). Generally, a DSC analysis involves increasing the sample holder temperature linearly with time and maintaining the reference and sample cells at the same temperature throughout the experiment. The differential power required to do this gives the required experimental data. Because there will be always be slight differences in volume and shape of the two cells, careful controls are required; the DSC experiment starts by loading buffer in each cell and collecting “buffer/buffer” runs. Protein is then loaded in the sample cell and “protein/buffer” runs are collected. Buffer/buffer runs are subtracted from the protein/buffer runs to account for cell differences.
The main application of DSC is measurement of macromolecular unfolding due to heat denaturation. Proteins undergo a transition, from a native, biologically active conformation (N) to a denatured conformation (D) with increasing temperature, a property they share with many other biological macromolecules e.g. DNA. The N and D states are in reversible equilibrium and the concentrations of [N] and [D] change with temperature. At any one temperature we can define the equilibrium constant, Keq = [D]/[N] or ∆G=-RT ln Keq. The temperature at which the concentrations of D and N are equal is defined as the midpoint of the transition or melting temperature, Tm. At the Tm temperature, which is a measure of a protein’s thermal stability, Keq= 1 and ∆G=0.
DSC measures the heat capacity (Cp) by scanning the temperature up or down. Cp is simply the amount of energy required to raise the sample temperature by 1 degree K; it is related to enthalpy by the integral of Cp over a range of temperatures that span the native and denatured states. Unfolding arises from the disruption of the numerous interactions that maintain the native protein structure. Disruption of these alters ∆H and this is detected by DSC. The unfolding process is usually endothermic so energy has to be provided to keep the temperature at the same value as that of the reference cell. As well as the heat capacity changes from the unfolding process, there are contributions to Cp from the N and D states of the protein. These can be estimated from the regions on either side of the transition peak. After correction for these effects the area under the curve is a measure of the excess energy required to denature the protein. If we know the concentration of the protein solution and the volume of the calorimeter cell, we can convert this energy to ∆H in Joules or Calories per mol of protein. In a single DSC experiment, we can thus determine the transition midpoint, Tm, the enthalpy (ΔH) and the heat capacity change (ΔCp) associated with unfolding. The observed unfolding temperature, Tm, can be sensitive to complex formation since this can change ∆G by influencing the ∆H and ∆S terms contributing to the unfolding transition. Usually ligand formation stabilises the protein resulting in an increase in the observed Tm.
The Malvern VP-Cap-DSC is a designed specifically for biological samples with capillary cells wrapped around a metal core to increase the instrument sensitivity and that make automation very simple. The instrument has an auto sampler that uses deep 96-well plates kept at a user specified temperature.
We recommend protein samples at 20 µM and 0.4 ml volume. 5mls of buffer is required for buffer/buffer runs as controls and to condition and clean the reference and sample cells.
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