I am trying to stabilize a protein with sucrose. for this, I treat the protein with different concentrations of sucrose (0 to 1M). fluorescence intensity increase with the concentration of sucrose. However, the apex peak shows a redshift from 344 nm to 355 nm.
does this indicate protein aggregation?
Fluorescent tryptophan appears to be more exposed to solvent as seen from the observed red shift. These conformational changes may be due to aggregation or just sucrose binding.
Unfolded proteins tend to aggregate sometimes. Aggregate formation can cause some of the chromophores to be buried and other ones to be exposed to the solvent. This explains the bidirectional shift and the occurrence of peaks at 313 nm and 344 nm in the emission spectra.
Tryptophans show red shifted emission as polarity of the solvent increases i.e., on moving from a hydrophobic environment to hydrophilic environment.
Your signal is clearly very weak to be able to see the prominent Raman peak. And the fluorescence out around 370-380nm cannot be protein fluorescence. I suspect you have some contaminant present in the EDTA that fluoresces. I suggest that you excite at 275nm which will pull the Raman peak back to 303nm and will increase the protein fluorescence signal.
Dear Solmaz Kamrani ,
Interesting observation. There are basically three things to take into account:
-As indicated by Mohamed Khedawy a red shift means the Trp is in a more hydrophilic environment (in your case most likely more exposed to the water phase), a blue shift means a more hydrophobic environment (in your case most likely more buried within the protein)
-In relation to this ‘normally’ a red shift gives rise to a lower fluorescence signal (because the quantum yield in a hydrophilic environment is lower) while a blue shift gives rise to a higher intensity
-Lower fluorescence can be caused by self-quenching (in your case most likely aggregation) the observed higher fluorescence might be caused by less aggregation (less self-quenching of Trp-Trp fluorescence)
Indeed, the role of sucrose in preventing protein-protein interactions is seen before, see for example:
Olsson, C., & Swenson, J. (2019). The role of disaccharides for protein–protein interactions–a SANS study. Molecular Physics, 117(22), 3408-3416.
See for more background about red/blue shifts and possible self-quenching the following paper (and references therein):
Article Tryptophan fluorescence study on the interaction of the sign...
Best regards.
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