I checked protein ligand interaction through Fluroscence, I keep my protein concentration constant with varying concentration of ligand at the small concentration 5uM the FLuroscence is peak is hyperchromic and at 10uM concentration the peak is very lower and at 20 uM there is slightly increase in peak above the 2nd peak with 10uM concentration and as the concentration increases upto 200uM the peak shift to back same on my protein peak why this happens?? And I changed the another ligand same things happen please suggest me why this happens or what I can do to rectify this problem??
Dear Abhilasha Thakur ,
Using the intrinsic Trp fluorescence can be quite complicated. As a result of ligand binding two extremes can happen: Exposure to a more hydrophilic environment leads to a decrease in fluorescence (and slight corresponding red shift) while a hydrophobic environment will lead to a blue shift and increase in Trp fluorescence.
Things that are important to explain your observations:
-Is there only one Trp present or are there more?
-How well did you ensure a proper buffer (and the ‘right’ pH)?
-Did you check the pH upon increasing the addition of ligand solution?
-Did you perform a volume correction (with more AMP solution you dilute more)?
Etc. etc. I don’t know how well you already characterized your protein focussing on the Trp fluorescence (pH dependence, effect of ionic strength, folded and unfolded state, effect of temperature etc.). You could consider using quenchers to get a bit more ‘grip’ on your understanding of your protein. For some inspiration have a look at the following papers (see enclosed files):
Elalaoui, A., Divita, G., Maury, G., IMBACH, J. L., & Goody, R. S. (1994). Intrinsic tryptophan fluorescence of bovine liver adenosine kinase, characterization of ligand binding sites and conformational changes. European journal of biochemistry, 221(2), 839-846.
(2006) Protein Fluorescence. In: Lakowicz J.R. (eds) Principles of Fluorescence Spectroscopy. Springer, Boston, MA. https://doi.org/10.1007/978-0-387-46312-4_16
Hope this helps you a bit further.
Best regards.
One more point to add to those of Rob Keller : The inner filter effect also has to be considered. This phenomenon is the absorbance of light by the ligand. The absorbance of 280 nm excitation light by the AMP concentrations you are using could be substantial, reducing the fluorescence. To test and correct for this, repeat the titration using N-acetyltryptophanamide as a substitute for the protein with which AMP will not interact.
Good addition made by Adam B Shapiro . Indeed, AMP absorbance and fluorescence (not entirely sure how strong) is something to take into account, see for more details about this (see also enclosed file):
Walaas, E. (1963). Fluorescence of adenine and inosine nucleotides. Acta Chemica Scandinavica, 17, 461-463.
Best regards.
AMP is not fluorescent. It has a maximum wavelength of UV absorption at 259 nm with an extinction coefficient of 15,400 M-1cm-1. The extinction coefficient at 280 nm is much lower (roughly 1500-2000 M-1cm-1), but could account for some decrease in protein fluorescence due to inner filter effect when present at a high concentration. If the fluorescence cuvette has a pathlength of 1 cm, the average excitation pathlength is 0.5 cm. If the AMP concentration is 200 µM and the extinction coefficient at 280 nm is 1500 M-1cm-1, then the absorbance is 0.15, which corresponds to a reduction of transmitted excitation light of 30%.
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