Measuring Protein Thermostability by Differential Scanning Calorimetry

George I. Makhatadze1

1 Texas Tech University, Lubbock
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 7.9
DOI:  10.1002/0471140864.ps0709s12
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Differential scanning calorimetry (DSC) the method of choice to study the conformational stability of biological macromolecules and proteins in particular. This unit presents step‐by‐step protocols for DSC, including sample preparation and interpretation of the results in the simplest cases as well as calibration of the apparatus and maintenance of DSC cells. Various general experimental considerations, including possible pitfalls and errors, are also discussed.

PDF or HTML at Wiley Online Library

Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Differential Scanning Calorimetry
  • Calibration of the DSC Instrument
  • Support Protocol 1: Electrical Calibration
  • Support Protocol 2: Temperature Calibration Using Hydrocarbon Capsules
  • Support Protocol 3: Temperature Calibration Using Lipid Suspensions
  • Support Protocol 4: Maintenance and Cleaning of DSC Cells
  • Commentary
  • Figures
PDF or HTML at Wiley Online Library


Basic Protocol 1: Differential Scanning Calorimetry

  • Protein solution, dialyzed (unit 4.4 & appendix 3B) and in dialysis bag (see )
  • Equilibration buffer (buffer solution used for the last dialysis; see for discussion of buffer selection)
  • Pasteur pipet or automatic pipettor
  • Spectrophotometer and quartz cuvettes of appropriate optical path length (depending on the expected protein concentration)
  • 2‐ml all‐glass syringe (e.g., Becton Dickinson 2‐cc Yale syringe)
  • Precut needle supplied with the DSC instrument
  • Differential scanning calorimeter (DSC), calibrated (see Suport Protocols protocol 21, protocol 32, and protocol 43)

Support Protocol 1: Electrical Calibration

  • Lipid: dipalmitoylphosphatidylcholine (DPPC) or distearoylphosphatidylcholine (DSPC)
  • 10 mM sodium phosphate buffer, pH 7.0 sodium phosphate buffer, pH 7.0 ( appendix 2E)
  • Vortex mixer
  • Water bath, 60°C

Support Protocol 2: Temperature Calibration Using Hydrocarbon Capsules

  • 1 M HCl or 1 M NaOH
  • Distilled water
  • Concentrated formic acid
  • 95% (190‐proof) ethanol
  • 2% (w/v) SDS, or other detergent
  • Concentrated nitric acid
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Biltonen, R.L. and Freire, E. 1978. Thermodynamic characterization of conformational states of biological macromolecules using differential scanning calorimetry. CRC Crit. Rev. Biochem. 5:85‐124.
   Ghosaini, L.R., Brown, A.M., and Sturtevant, J.M. 1988. Scanning calorimetric study of the thermal unfolding of catabolite activator protein from Escherichia coli in the absence and presence of cyclic mononucleotides. Biochemistry 27:5257‐5261.
   Gill, S.C. and von Hippel, P.H. 1989. Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182:319‐326.
   Izatt, R.M. and Christensen, J.J. 1976. Heats of proton ionization, pK, and related thermodynamic quantities. In CRC Handbook of Biochemistry and Molecular Biology, Vol. 1: Physical and Chemical Data (G.D. Fasman, ed.) pp. 151‐269. CRC Press, Cleveland.
   Jaenicke, L. 1974. A rapid micromethod for the determination of nitrogen and phosphate in biological material. Anal. Biochem. 61:623‐627.
   Kidokoro, S. and Wada, A. 1987. Determination of thermodynamic functions from scanning calorimetry data. Biopolymers 26:213‐229.
   Krishinan, K.S. and Brandts, J.F. 1978. Scanning calorimetry. Methods Enzymol. 49:3‐14.
   Mabrey, S. and Sturtevant, J.M. 1976. Investigation of phase transitions of lipids and lipid mixtures by sensitivity differential scanning calorimetry. Proc. Natl. Acad. Sci. U.S.A. 73:3862‐3866.
   Makhatadze, G.I. 1998. Heat capacities of amino acids, peptides and proteins. Biophys. Chem. 71:1‐26.
   Makhatadze, G.I., Medvedkin, V.N., and Privalov, P.L. 1990. Partial molar volumes of polypeptides and their constituent groups in aqueous solution over a broad temperature range. Biopolymers 30:1001‐1010.
   Marky, L.A. and Breslauer, K.J. 1987. Calculating thermodynamic data for transitions of any molecularity from equilibrium melting curves. Biopolymers 26:1601‐1620.
   Pace, C.N., Vajdos, F., Fee, L., Grimsley, G., and Gray, T. 1995. How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 4:2411‐2423.
   Plotnikov, V.V., Brandts, J.M., Lin, L.N., and Brandts, J.F. 1997. A new ultrasensitive scanning calorimeter. Anal. Biochem. 250:237‐244.
   Privalov, G., Kavina, V., Freire, E., and Privalov, P.L. 1995. Precise scanning calorimeter for studying thermal properties of biological macromolecules in dilute solution. Anal. Biochem. 232:79‐85.
   Privalov, P.L. and Potekhin, S.A. 1986. Scanning microcalorimetry in studying temperature‐induced changes in proteins. Methods Enzymol. 131:4‐51.
   Privalov, P.L. and Khechinashvili, N.N. 1974. A thermodynamic approach to the problem of stabilization of globular protein structure: A calorimetric study. J. Mol. Biol. 86:665‐684.
   Sanchez‐Ruiz, J.M. 1992. Theoretical analysis of Lumry‐Eyring models in differential scanning calorimetry. Biophys. J. 61:921‐935.
   Scopes, R.K. 1974. Measurement of protein by spectrophotometry at 205 nm. Anal. Biochem. 59:277‐282.
   Sturtevant, J.M. 1972. Calorimetry. Methods Enzymol. 26:227‐253.
   Winder, A.F. and Gent, W.L. 1971. Correction of light‐scattering errors in spectrophotometric protein determinations. Biopolymers 10:1243‐1251.
   Wintrode, P.L., Makhatadze, G.I., and Privalov, P.L. 1994. Thermodynamics of ubiquitin unfolding. Proteins 18:246‐253.
   Yu, Y., Makhatadze, G.I., Pace, C.N., and Privalov, P.L. 1994. Energetics of ribonuclease T1 structure. Biochemistry 33:3312‐3319.
Key References
   Biltonen and Freire, 1978. See above.
  Three references that discuss in detail the theoretical background for deconvolution of the heat capacity profile, each describing a separate approach to data analysis.
   Privalov and Potekhin, 1986. See above.
  Basic reference containing tables of heats of ionizations and other thermodynamic information for different buffer systems.
   Kidokoro and Wada, 1987. See above.
  Detailed discussion on the calculation of protein extinction coefficients from amino acid compositions.
   Izatt and Christensen, 1976. See above.
  Three reviews describing and assessing the determination and interpretation of protein stability by DSC.
   Pace et al., 1995. See above.
  Detailed discussion of the analysis of irreversible denaturation processes.
   Privalov, P.L. 1979. Stability of proteins: Small globular proteins. Adv. Protein Chem. 33:167‐241.
   Privalov, P.L. 1982. Stability of proteins: Proteins which do not present a single cooperative system. Adv. Protein. Chem. 35:1‐104.
   Makhatadze, G.I. and Privalov, P.L. 1995. Energetics of protein structure. Adv. Protein. Chem. 47:307‐425.
   Sanchez‐Ruiz, 1992. See above.
PDF or HTML at Wiley Online Library