Prometheus records fluorescence data (nanoDSF) at 330 nm and 350 nm and automatically calculates the F350/F330 ratio. When a globular protein unfolds, typically tryptophan groups buried in the hydrophobic core are exposed to the solvent, yielding a characteristic increase of the F350/F330 ratio (red shift). However, not all proteins follow this pattern and the unfolding event can also result in a decrease of the F350/F330 ratio (blue shift).

Occasionally, there can already be a decrease in the F350/F330 ratio at low temperatures which would not correspond to an observable transition in a DSC thermogram, when measuring the same sample with e.g. Differential Scanning Calorimetry (DSC). Thus, the temperature change at low temperatures does not cause a measurable heat change but rather leads to a subtle conformational change or a solvent rearrangement which affects the tryptophan groups and has a significant spectroscopic effect but a negligible thermal effect.

The F350/F330 ratio is dominated by tryptophan but also contains contributions from tyrosines. Consequently, the observed signal is a weighted average corresponding to the spectral micro-environments of all tryptophans and tyrosines in the sequence. Thermally induced unfolding often is much more complicated than a simple two-state model of folded and unfolded protein would suggest. Therefore, the observation of such an “anomalous” spectroscopic profile upon heating does not necessarily mean that the protein is very unstable.