I am studying conformational change in a peptide and monitored λmax after exciting it at 295 nm. The peptide was dissolved in 20mM glycine HCl buffer (pH 2.4) and it contains single tryptophan. At t=0, λmax was 360 nm. With time, it further red shifted and observed λmax was 365 nm. How is it possible, considering fully exposed tryptophans have emission maximums at 355 nm? Note that peptide underwent amyloidogenic transformation.
The lambdamax of Trp is highly dependent on the solvent polarity. To me it doesn´t sound unusual that you get a lamdbamax of 365nm. You should take some L-Trp (or better N-Acetyl-Tryptophanamide) and measure lambdamax of it in your buffer system. That will give you an answer what the actual lambdamax of fully exposed Trp in your system is. Concerning the drifting lambdamax... Your buffer conditions are pretty acidic. Perhaps your Amyloids are dissolving under these conditions and you actually expose Trp-residues to the solvent due to loss of structure? How long do you incubate your amyloid peptide in the glycine ph2 before you do the measurement? Are they sufficiently equilibrated?
Is the Raman-peak always at a wavelength of ~325-330nm upon excitation at 295nm? This would exclude any issues with the instrument.
Best regards.
Probably a calibration issue or a spectral correction issue. Anyway, check papers by Callis (published a lot of stuff on the subject) to have a full description of the possible changes of Trp fluorescence as a function of the environment. (Other effects aside from solvation can shift the emission peak..)
Various cyclic peptides with one try have shown fluorescence maximum up to 359 nm, suggesting that the different tryptophan rotamers have different emission maxima. Molecular dynamics simulations support this occurrence for the six canonical side chain rotamers of solvated tryptophan for which an emission max up to 365 nm has been calculated. You could also consider the presence of non-tryptophan fluorescence (a different fluorophore). However, it is best to use as a reference N-Acetyl-Tryptophanamide using the same solution of the protein as suggested by Alexander Tischer.
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