or is it simply not comparable at all? since fluorescence intensity decreases strongly with temperature and increases (slightly) with GdnCl concentration.

is the effect of the temperature due to higher quenching at higher temperatures (thereby decreasing fluorescence life time?)

There is a temperature effect for sure and perhaps you have to take in account other technical issues, compare the absorption signal, check if you have protein aggregation etc. In general, it is not easy to directly compare protein unfolding in different conditions, for several reasons, you have to take in account that partial unfolding may occur in different regions of the protein causing different fluorescence variations. If I remember correctly lysozyme has multiple trp located in different areas whose signal can be affected in different ways from local environment. However, when protein unfold in some case you may find large increases in their intensities while in other instances you find a decrease upon unfolding of the chain. Usually in a hydrophobic environment (buried within the core of the protein), these residues show a high quantum yield and therefore a high fluorescence intensity, if exposed to solvent their quantum yield decreases leading to low fluorescence intensity and a red shift is observed when Tryptophans are exposed to a more polar environment, you can obtain different results in dependence of tryptophans environment.

Thanks a lot Valeria. It's a very good point that the trp's are located in different areas and therefore responds differently to denaturation.

I just found last night that the temperature effect (on internal conversion) is enormous and is the explanation for the decrease in fluorescence intensity upon heat denaturation.

Right now, what puzzles me the most is the question why the fluorescence increases upon chemical denaturation. As you said, quantum yield of exposed trp is generally lower. But how does this fit together?

Is this rather an effect of the absence of internal quenchers in the unfolded state?

you shouldn't be surpise if you found fluorescence increase, the key point is that it depends on the trp environment and on conformational changes occurring during unfolding. With fluorescence there is never a general rule. Anyway better you check if you have aggregation (e.g. simply looking at the elastic scattering peak), check if you have significant changes in absorption spectrum and verify all possible experimental issues. Then i'm pretty sure that lysozyme chemical denaturation is widely studied in literature, with multiple experimental techniques, try to take a look to previously pubblished papers may be there is an established explanation.

Dear Dominik, can you suggest the refs on  the temperature effect on internal conversion and  the decrease in fluorescence intensity upon heat denaturation?