The dissociation constant (Kd) for the protein-nanoparticle complex (in terms of the nanoparticle concentration) can be obtained by plotting the fluorescence intensity (y) versus the nanoparticle concentration (x), then fitting the data to a binding equation, for example y = (a)(x)/(Kd + x), using nonlinear regression, where a is the intensity at saturation.

Make sure to subtract any background fluorescence intensity from the nanoparticles by repeating the titration without adding the protein.

The increase in fluorescence intensity is possibly caused by the fluorophore becoming protected from solvent quenching by its interaction with the surface of the nanoparticles.

Your nanoparticles has any fluorescence properties?

If fluorescence intensity increasing after addition of nanoparticles, this process is (Fluorescence enhancement effect). In this case may be your protein molecules aromatic residues is more exposed because of alteration in secondary structure of protein with nanoparticles.

Reference book.

Principles of Fluorescence Spectroscopy: Joseph R Lakowicz

Thank you Bipin S Khade sir for your reply..

The nanoparticles used in the experiment do not have any fluorescent properties..

Regarding alteration in secondary structure, it is getting stabilized in presence of these nanoparticles as confirmed from CD spectroscopy

What is the size of your nanoparticle? Can you please show the absorption spectra of the nanoparticle and the fluorescence spectra of the protein with addition of nanoparticle? In order to test the stabilization/exposure/conformational change you may check the lifetime of the fluorophore. As you know (assuming that the radiative rate constant remains same) quantum yield and lifetime is proportional.

The dissociation constant (Kd) for the protein-nanoparticle complex (in terms of the nanoparticle concentration) can be obtained by plotting the fluorescence intensity (y) versus the nanoparticle concentration (x), then fitting the data to a binding equation, for example y = (a)(x)/(Kd + x), using nonlinear regression, where a is the intensity at saturation.

Make sure to subtract any background fluorescence intensity from the nanoparticles by repeating the titration without adding the protein.

The increase in fluorescence intensity is possibly caused by the fluorophore becoming protected from solvent quenching by its interaction with the surface of the nanoparticles.

Thank u Adam B Shapiro sir...I will do it and if I have any doubts i will ask you again..

Thanks once again

Thanks Tuhin khan sir..

1. One nanoparticle is 18nm..other one is 24nm

I want to know what type of nano-particles which you are using.

Imocha rajkumar..I used hydroxyapatite and zinc oxide nanoparticles

If the nanoparticles, at the concentrations undertaken give a subtantial UV-vis absorbance at the excitation or emission wavelengths of the protein fluorescence, go for inner filter effect corrections. If the values of correction factors are close to 1, you can ignore the inner filter effect.

Then calculate the binding constant (Kb)using Benesi-Hildebrand (B-H) equation. For calculating quenching constant (Ksv), Ge et al. have used Stern-Volmer equation, even when quenching was followed by an increase in fluorescence intensity, (Langmuir 2012, 28, 5913−5920). You can use either of the equations you find suitable. Following reference will be very helpful for you:

Ind. Eng. Chem. Res. 2017, 56, 13663−13676.

If the protein is hemoglobin or myoglobin, the increase in fluorescence intensity may be explained as: "As a consequence of all these noncovalent interactions, the distance between the Trp and heme increases, which can depress the quenching reaction between them, causing the fluorescence intensity to increase significantly." (Ind. Eng. Chem. Res. 2017, 56, 13663−13676)