The Prometheus records fluorescence data at 330 nm and 350 nm, and automatically calculates the F350/F330 ratio. The different detection modes can yield different results.

In the single wavelength detection, we look at the change in fluorescence intensity at a defined wavelength over temperature. The fluorescence ratio F350/F330 however is a measure for a spectral shift in the emission profile of the Tryptophan (Trp) residues

Since the 350 nm/330 nm ratio typically obliterates fluorescence effects of autofluorescent additives by specifically detecting tryptophan-specific fluorescence emission shifts, this detection mode is typically more robust than single wavelength detection. It also obliterates the exponential decay of fluorescence intensity with increasing temperature, since the temperature-dependence of fluorescence affects both wavelengths equally. Therefore, clear inflection points (and Tms) can be derived which might not be visible in the single wavelength detection mode.

Vice versa, it is also possible that unfolding events which do not trigger an emission peak shift are visible in the single wavelength data, but not in the ratio. It is therefore recommended to inspect all the obtained data, as important structural information might be obtained by comparing the unfolding profiles in the three analysis modes.

There are two different possible explanations why the melting points are different:

1.The IPs/melting points obtained from the different analysis modes deviate slightly (typically 1-2 °C):

As the fluorescence of Trp and Tyr exponentially decreases with temperature, we calculate the ratio between the two wavelengths to correct for baseline drifts. This mathematical operation tilts the resulting curve as compared to the single wavelengths, resulting in a slightly deviating transition point. This procedure however yields most reproducible data and provides further insight into structural details by revealing spectral shifts of fluorescence emission. In such a case the melting point obtained by the 350 nm/330 nm ratio is preferably used.

2. The IPs/melting points obtained from the different analysis modes deviate more than 2 °C:

In this case it is likely that the two melting points reflect two distinct events (e.g. unfolding of two different domains). If Trp residues are already surface-exposed in the native state, an unfolding event might not necessarily result in change of emission wavelength (typically a red-shift). This can cause an unfolding transition point in the single wavelength analysis but not in the ratio analysis. The melting point you see in the ratio analysis is likely the result of the unfolding of a different part of the protein structure (e.g. a different domain) where Trp residues are buried in the hydrophobic core of the protein and show a red-shift of their emission spectra upon unfolding.