Joyce Johnson

Technical Note: Measurement of Lysozyme Gibbs Free Energy via Chemical Denaturation

Chemical denaturation is a powerful technique for assessing proteins’ stability and can help inform decisions about which antibody construct or formulation to develop further. In this technical note, the Gibbs Free Energy and the midpoint of inflection values were measured for a lysozyme sample. The lysozyme sample was denatured using the chaotropic agent guanidine hydrochloride, and the change in the intrinsic spectrum was measured with the SUPR-CM. The Gibbs Free Energy and the midpoint of inflection values were determined by fitting to the full denaturation curve using a two‑state function.


  • The Gibbs Free Energy of unfolding (∆G°) and midpoint of inflection denaturation concentration (Cm) are measured for the globular protein lysozyme (acetate buffer, pH 5.2)
  • Chemical denaturation was used to unfold the protein and the change in fluorescence spectrum was measured with the SUPR-CM fluorescence plate reader
  • The ratio of intensities at 354 nm and 338 nm was used to chart the unfolding process as the denaturant concentration was increased
  • Fitting of a two-state function produced a Gibbs Free Energy and midpoint of inflection values with low errors and that align with literature values.

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Technical Note