Interaction between free Trp in solution and TNS

Repeat the experiment between TNS and BSA by replacing BSA with free L-Trp. What do youobserve By plotting I0/I vs. [TNS] what is the nature of the constant you will calculate  

The decrease in fluorescence intensity of the Trp residues is the result of energy transfer that is occurring from aromatic residues to bound TNS. The fluorescence of the ligand increases as a result of its binding to BSA and of the energy transfer that is occurring from Trp residues to TNS. 

The inverse plot of a hyperbolic equation is linear, and so the inverse plot of Equation (15.1) is 

Plotting 1/A/ vs. 1/[TNS] yields a straight line with a slope equal to fCd/AImax and intercept equal to 1/A/mlx. 

Application of Equations (15.2) and (15.3) in the determination of the dissociation constant of a complex implies that at every concentration of ligand we add, most of the ligand is bound to the protein, and so there is a small or negligible concentration of free ligand. When this is not the case, the application of Equations (15.2) and (15.3) is not appropriate, and another equation should be derived and applied to take into consideration the concentration of free ligand. Let us develop this equation. Binding of a ligand L to a protein P can be written as follows  

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