Record an excitation spectrum with emission max at 340 nm and scan range 200-330 nm. You won't see the 230 nm peak. 230 nm band doesn't give you information about the tryptophan or tyrosine.

280 nm spectra is for the transition of diffused pi electrons of the aromatic ring of try or trp. Because the excitation of the conjugated pi electrons requires lower energy. The high energy absorption at 230 nm or below is due to the n->pi* or pi->pi* transitions of the carboxylic group or amide in protein.

Do not rely on the absorption below 250 nm if the experiment was done in aerial condition. Dissolved oxygen must be removed from the sample by passing nitrogen or inert gas through the sample and the spectra should also be recorded in continuous nitrogen flowing condition if you want to report the below 250 nm spectra. As in circular dichroism experiments where we record the differential absorption at far UV regions. Oxygen absorbs heavily at this range.

I think the absorption at 230nm is account for phenolic ring in Trp and at 280 for the whole aromatic moiety (indole ring). This can happen for Tyr.

The short answer is 'energy transfer'. In the same way, we know that the absorption/excitation band of NADH gives emission at 450nm, but you can also excite that emission with 260nm light which is absorbed initially by the purine ring of NADH, The energy is transferred between the two chromophores.

Because of the ring structure in the functional groups of these aminoacids (residues Phe, Trp and Tyr), which enables the resonating electrons to reach higher energy states upon excitation with light.

and thanks Stéphane Abel for the link.. very useful..

Because of the ring structure in the functional groups of these aminoacids (residues Phe, Trp and Tyr), which enables the resonating electrons to reach higher energy states upon excitation with light.

and thanks Stéphane Abel for the link.. very useful..

Dear Vanessa,

This is due to different electron transitions: the excitation correlates with the absorption of tryptophan at ca. 225 nm and ca. 270 nm which correspond with special electron transitions. For details look to the review papers of David Creed in Photochemistry and Photobiology 39 (4), (1984), 537-562 - review on tryptophan, and: David Creed in Photochemistry and Photobiology 39(4), (1984), 563-575 - review on tyrosine.

Karola Schaefer

Record an excitation spectrum with emission max at 340 nm and scan range 200-330 nm. You won't see the 230 nm peak. 230 nm band doesn't give you information about the tryptophan or tyrosine.

280 nm spectra is for the transition of diffused pi electrons of the aromatic ring of try or trp. Because the excitation of the conjugated pi electrons requires lower energy. The high energy absorption at 230 nm or below is due to the n->pi* or pi->pi* transitions of the carboxylic group or amide in protein.

Do not rely on the absorption below 250 nm if the experiment was done in aerial condition. Dissolved oxygen must be removed from the sample by passing nitrogen or inert gas through the sample and the spectra should also be recorded in continuous nitrogen flowing condition if you want to report the below 250 nm spectra. As in circular dichroism experiments where we record the differential absorption at far UV regions. Oxygen absorbs heavily at this range.

Thank you very much for all your comments. Now I have a better understanding of my analysis.

Hi Vanessa,

Sorry for my english bad , but i will try.

This peak in 230 nm is for side chain of triptophan, he is characteristic of interaction between aromatic group of side chain.

This peak is common in protein that have B-sheet its like lyzozyme,

I wait help have you,

Basically, the lower wavelength (210 to 230 nm) is because of the peptide bond as stated above and 270 to 280 is because of the aromatic group. I think, its absolutely true.... 

But, one more question is why the shape of tryptophan absorbance looks like having three different peaks where as phenyl alanine and tyrosine are comparatively are having single peaks?

To my question, I have an answer but I do not know how far it is justified!!!! The branching of absorbance peak (271, 280 and 295) for Trp is because of its local conformation. The rotation of the groups around the carbon-carbon single bond.....