hello
kind regards
I study the structure and activity of the digestive enzymes. The enzyme I studied contains 5 amino acids tryptophan. In one of the studies, I study the effects of a food dye on the enzyme structure. How can I check if the enzyme-dye complex is fluorescent or non-fluorescent?
You can observe native fluorescence in fluorimetry. You can very well study the Trp (and Phe but its like 10x lower) and will see how the fluorescence changes with the dye present or under different conditions. You will for sure see changes and experienced biophysicist will help you explain the structural reasons for them
Dear Elham Yadollahi ,
This can be quite challenge. It all depends on the food dye you want/need to use.
But first you need to characterize your enzyme. As said by Marek Zarzycki it is known that tryptophan itself has fluorescence features that has extensively been used to study the structure of proteins, see for an excellent overview:
Vivian, J. T., & Callis, P. R. (2001). Mechanisms of tryptophan fluorescence shifts in proteins. Biophysical journal, 80(5), 2093-2109.
https://www.sciencedirect.com/science/article/pii/S0006349501761838
Once this has been performed fully, it depends as said on the food dye you want to use, see for a nice example:
Zhang, G., & Ma, Y. (2013). Mechanistic and conformational studies on the interaction of food dye amaranth with human serum albumin by multispectroscopic methods. Food chemistry, 136(2), 442-449. (in Google Scholar you find a free version).
Or
Liu, B. S., Wang, J., Xue, C. L., Yang, C., & Lü, Y. K. (2011). Spectroscopic studies on the interaction of synthetic food colorants with bovine serum albumin. Zeitschrift für Physikalische Chemie, 225(4), 455-468. (see enclosed file)
Hope this helps you further.
Best regards.
You will use a spectrofluorometer and a quartz fluorometer cuvette for the protein sample. The concentration of the protein sample should be at least 1 micromolar. Set the excitation wavelength to 290 nm to specifically excite the tryptophan residues. Perform an emission wavelength scan to identify the peak emission wavelength, which will be between 330 and 360 nm, depending on the environments of the Trp residues.
Next, repeat the same scan, replacing the protein sample with the buffer that as used but no protein. This will give you the background signal, which is caused by Raman scattering from water. Subtract the buffer spectrum from the protein spectrum to see the spectrum of the protein itself.
When you add the dye, things get complicated. The dye may be fluorescent itself, so you should also measure its fluorescence in the absence of the protein, using exactly the same conditions as for the protein, including all the instrument settings. If the dye is fluorescent, you will have to subtract its fluorescence from the total dye + protein fluorescence.
Depending on the dye concentration, you may also have to concern yourself with the inner filter effect, which is caused by the absorbance of the dye. By absorbing light entering or leaving the sample, the dye will reduce the fluorescence of the protein. This effect can be measured by replacing the protein with N-acetyl-L-tryptophanamide (NATA) as the source of Trp fluorescence, since it is assumed that the dye does not bind to this compound. A correction can be made for the inner filter effect using the effect of the dye on the NATA fluorescence.
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