The absorbance of aromatic amino acids changes upon unfolding or refolding. However these changes are rather small compared with other observables obtained by CD, Fluorescence or DSC. You may want to look deeper into the topic of 2nd derivative UV-spectroscopy (e.g. http://www.ncbi.nlm.nih.gov/pubmed/?term=2nd+derivative+spectroscopy+protein+folding or http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1978.tb12622.x/pdf). This technique would enable you to follow folding or unfolding processes. Please note that you need the complete absorption spectrum but not a specific wavelength.

Cheers.

The absorbance of aromatic amino acids changes upon unfolding or refolding. However these changes are rather small compared with other observables obtained by CD, Fluorescence or DSC. You may want to look deeper into the topic of 2nd derivative UV-spectroscopy (e.g. http://www.ncbi.nlm.nih.gov/pubmed/?term=2nd+derivative+spectroscopy+protein+folding or http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1978.tb12622.x/pdf). This technique would enable you to follow folding or unfolding processes. Please note that you need the complete absorption spectrum but not a specific wavelength.

Cheers.

If you do not have metal ions or other cofactors that would function as chromophores, then the near UV portion of the spectrum will be of primary interest to you. All three aromatic amino acid side chains (tryptophan, tyrosine, and phenylalanine) and cysteine absorb between 250 and 320 nm. Since tryptophan and tyrosine having prominent absorption bands between 275 and 285 nm, I would begin looking at this spectral region for changes in absorbance during folding or unfolding.

Alexander is correct. The changes in absorption you would be observing will be small and second derivative spectroscopy is very viable option, and probably the best approach. With that said, a zero-order spectral analysis is possible. When proteins are in unfolded states their UV spectra usually shift to lower wavelengths due to the increased polarity of of the surrounding medium, if you monitor the absorbance along the edge of an absorption band (a region of maximal slope, say around 290 nm) you can follow the unfolding process.

Best regards

John

I'd be worried about looking at protein folding using UV-Vis. Too non-specific. What if it misfolds into aggregates after you decrease the urea concentration? (e.g. inclusion bodies which may have a similar b-sheet structure as amyloid). It will be "folded" but not native. Unless you have some specific assay (e.g. enzyme activity), CD or 1D NMR (look for high field methyls compared to a standard sample) would be much better than UV-Vis. Maybe even native-gels.

A protein derived from inclusion body often has a high tendency to aggregate. But, if it aggregates, fluorescence, far UV CD, and other spectroscopic techniques fail because the solution becomes opalescent and prevents the measures due to the high light scattering). In these cases the second derivative spectroscopy is optimal because any background which increases linearly (as opalescence, which increases linearly towards the blue with a high diffuse light) is canceled in the second derivative. So you see only the chromophors of interest. A gel electrophoresis will tell us (before the measurement) if the protein has a tendency to aggregate. The advantage of the technique is that it is non-destructive, it's cheap and there are many applications in the literature.

Alright, I think I will go for the secondary derivative spectroscopy and CD spectroscopy for protein folding analysis. Thanks everyone for the valuable comments!