DSC instrument design and corresponding experimental methodology

1. DSC instrument design and corresponding experimental methodology

2. The technical challenge with DSC • In DSC we are attempting to determine the heat capacity of a biomolecule, in aqueous solution, as a function of temperature • A typical small protein at 1.0 mg/ml has a molar concentration of ~0.04 mM • Water will be present at 55 M • approximately 6 orders of magnitude greater concentration • Water is unusual in that it has a high heat capacity (due to extensive H-bonding interactions with neighboring waters) How can the heat capacity of the protein (small signal) be distinguished from the contribution of solvent (large signal)?

3. Differential Scanning Calorimeter • The DSC contains two sample cells • One cell contains biomolecule (e.g. protein) in buffer (solvent) • The other cell contains only the buffer • In principle, subtraction of the heat capacity of the buffer sample from the biomolecule sample results in the heat capacity contribution of the biomolecule alone (this is the “differential” part of the DSC) • DSC cells are either capillary or “lollipop” in shape, and there are always two of them: “sample” cell “reference” cell

4. The DSC cells are contained in an insulated “adiabatic” chamber • Heaters are used to transfer heat energy to the cells (to raise the temperature) • The differential power required to maintain the heating rate of the sample cell, in comparison to the reference cell, is the data that is collected in an experiment “sample” cell “reference” cell Insulated adiabatic chamber Heater Heater Microcontroller Power (watts) Differential Cp(T)

5. Another technical issue • Although the two cells in the DSC are manufactured to be as identical as possible, there will be slight differences in volume, shape, etc • A difference in volume will result in a difference in overall heat capacity (more volume, greater heat capacity) • A difference in shape (and related physical differences) results in different Cp(T) properties • The magnitude of these differences can be substantially greater than the magnitude of the protein heat capacity (Cp(T)) How can the differences in the two cells be accounted for? (It is not practical to try to manufacture them exactly the same)

6. DSC data for both cells filled with buffer: Perfectly matched cells The typical situation Cp(T) Cp(T) 0 0 Temp (T) Temp (T)

7. Resulting DSC data for a protein sample: The typical situation Perfectly matched cells Cp(T) Cp(T) 0 0 Temp (T) Temp (T)

8. Differences between cells can be accounted for by subtracting a “buffer/buffer” run from a “protein/buffer” run: Protein/buffer data Buffer/buffer data Protein data - = Cp(T) 0 0 0 Temp (T) Temp (T) Temp (T) • The DSC experiment starts by loading buffer in each cell and collecting “buffer/buffer” runs • Protein is 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

9. The DSC has a “thermal history” • Consistent DSC scans are obtained only after the instrument has gone through at least one (likely two) cycles of heating and cooling • Any thermal variations will result in an aberrant scan • Stopping/starting scan process • Introducing a sample that is at a different temperature than the DSC cell • The protein sample is introduced only: • After several cycles of heating/cooling (with buffer/buffer runs) have been completed • With the protein at the same temperature as the DSC cell (typically 25 °C) • Without pausing or interruption of the instruments heating/cooling cycle

10. Practical experimental design: • Both cells are loaded with buffer • The instrument is setup for multiple (20) data collection runs (heating/cooling cycles) • “Buffer/buffer” data is collected (≥3 runs) • When the instrument is cooling down, prior to a heating cycle, the protein is introduced at 25 °C • A “protein/buffer” data run is collected • Similar protein loading is repeated two more times to obtain 3 “protein/buffer” scans

11. DSC Heating/cooling cycles and protein sample application: Post-run cooling Post-run cooling Heating cycle #1 Heating cycle #2 Temp 25°C (time) • Remove buffer from reference cell and fill with protein sample at this point in the post-run cooling • Do not interrupt heating/cooling cycles (i.e. don’t stop/start runs) • Have protein at room temperature (25 °C) (Note: here we are looking at the instrument heating/cooling cycles and not the heat capacity data)

12. Air bubbles are a real problem in DSC data collection • Air bubbles displace liquid and therefore reduce the heat capacity (yielding erroneous results) • Air bubbles can dissolve into solution over time • There will be an aberrant increase in heat capacity each subsequent cycle as the bubble dissolves • To address this issue: • Samples & buffer are degassed (10 min) • DSC cell is kept under pressure (~35 psi) • A certain technique is used in filling the cells

13. How to tell if an air bubble is present: • Buffer/buffer scans will be inconsistent • Heat capacity increases in subsequent scans as bubble dissolves Buffer/buffer data #4 Cp(T) #3 #2 Scan number #1 Temp (T)

14. Pressure changes affect the apparent heat capacity • Opening/reclosing the cell is necessary to introduce the protein sample • The pressure is never exactly the same after replacing the pressure cap • This small pressure change results in an effective Y-offset to the Cp(T) data: Protein data Pressure 1 Pressure 2 Cp(T) Pressure 3 Temp (T) This is not a critical issue for the derived thermodynamic parameters