ANS binds to all sorts of hydrophobic surfaces. I would suggest you do control experiment: Titrate Safranal into the ANS solution in absence of the protein. Make sure that ANS and Safranal do not interact with each other. Also measure spectra and not only a single wavelength. You might also learn something from that. At which wavelength have you measured the ANS-Fluorescence?

Cheers

Dear Alexander

Thanks for your comment. I have done the control experiment and didn't observe any significant interactions; however, I subtracted the control results from the obtained ones. The excitation wavelength was 380 nm and the emission was recorded from 450 to 650 nm and the max intensity was reported in the displayed curve.

Kind regards

Dear Ali,

Are these results with substracted control (safranal and ANS without protein)? If they are, sometimes its difficult to receive reliable data for very low concentrations, as 0.05 microM. After this point you received dose dependent decrease in ANS fluorescence. This could be due to some conformational changes within tubulin molecule. But it is not sure if they are due to safranal interaction or ANS interaction to tubulin. ANS fluorescence is very sensitive method, but in my oppinion you need some additional experiments: to titrate protein-safranal solution with different ANS concentrattions and to measure the changes in ANS fluorescence. This could give you more details about your investigation of tubulin hydrophobicity. 

Kind regards

Dear Lidiya

Yes, this is the result with subtracted control. Actually, the intrinsic fluorescence of tubulin titrated by various concentrations of safranal was recorded. In this method, the solution was excited at 295 nm and the emission spectra of tryptophan residues were drawn between 300-450 nm. You can find the relative result in the attached file. The fluorescence intensity reduced in a dose-dependent manner.

I'd be grateful if let me know your own opinion about observed results. The conformational changes of tubulin in the presence of safranal are clear and observable. I ask you to interpret these changes and tell me what you can find out these results.

Truly yours

Dear Ali,

The changes in the fluorescence intensity of Trp fluorescence in the presence of safranal are really clearly visible. These results could be due to the interaction between tubulin and safranal, but you may analyse your data with some statistical program that will give additional information about this interaction. 

If I understand corectly, the first picture you upload shows the changes of ANS fluorescence upon titration of tubilin with sarfranal? If it is true, it means that changes coud be really either due to safranal binding to tubulin (indirect evidence by means of ANS fluorescence) or due to ANS bunding to tubulin. (It is possible the two ligands ANS and safranal to bind tubilin).

I think it is a good idea to make additional competitive experiment: titration of protein-safranal solution with different ANS concentrattions and to measure the changes in ANS fluorescence. This will give you more detailed picture of you results that will give you certainty to interpete them with convivtion.

Kind regards,

Lidiya 

Dear Lidiya

Many thanks for your kind guidance.

Best Regards