I' believe this might be energy transfer from Trp to NAD or NADPH (excitation 340-360 emission ~460 nm).

First be sure that it was tryptophan by exciting it at 295 nm. At this wavelength the extinction coefficient of tyrosine is very low and specifically Trp gets excited.

You should also check the Raman scattering peak position to be sure that the instrument calibration is in order. When you are exciting at 280 nm you should get a Raman scattering peak at 309 nm and while exciting at 295 nm it should be at 328 nm.

Are you talking about more than 100 nm red shift! First make sure that the cuvette was clean. It's unusually high Stokes' shift for tryptophan. The attached article might be helpful.

http://www.pnas.org/content/105/42/16095.full

Thank you very much for your help. In fact, in the spectra I see the normal emission of tryptophan and this emission at 280/460-470. There is a compound that emits at this value, Diamino Phenyl Oxydiazole (DAO), but I'm not sure how this could be related to proteins.

Do you know whether azatryptophans can occur naturally in proteins? because in the paper they add this compound to cause fluorescence

azatryptophans can be naturally incorporated into proteins in place of tryptophan if you add them to the cell culture. That's a very neat way of introducing them. Take a look st: De Filippis, V.; De Boni, S.; De Dea, E.; Dalzoppo, D.; Grandi, C.; Fontana, A. Protein Sci. 2004, 13, 1489–1502, and Twine, S. M.; Szabo, A. G. Methods Enzymol. 2003, 360, 104–127.

On the other hand, tryptophan emission can indeed change depending on the environment, that's widely known, but changes are never as much as 100 nm.

Did you consider energy transfer from tryptophan to some other chromophore not related to proteins? This could explain why you see normal tryptophan emission and emission at 460-470 nm.

I' believe this might be energy transfer from Trp to NAD or NADPH (excitation 340-360 emission ~460 nm).

Hello. I've found a paper about that peak. It seems to be the binding of NADH to an enzyme, and energy transfer from tryptophan in the enzyme to NADH.

This is very likely a key to your question. NAD (and NADP) are not intrinsic protein fluorophores, but they occur as co-enzymes in redox reactions.