What is fluorescent in your experiment? The fluorescent substance should be kept at a fixed concentration, and the non-fluorescent substance should be titrated. The change in fluorescence (y-axis) should be plotted against the titrant concentration (x-axis). The resulting curve should be fit to a suitable binding equation. This will give you the Kd of the equilibrium. It can also reveal whether there is cooperative binding. This analysis is simplest when the concentration of the fluorescent substance is well below the Kd.

To measure the stoichiometry of binding (ligand molecules per base pair), the titration should be done at a high concentration of ligand relative to the Kd (tight binding regime). Then the amount of DNA necessary to get maximal binding can be compared to the concentration of the ligand to get the stoichiometry.

@Adam,

Thank you for your reply. In my experiment I have titrated the DNA against the fixed concentration of ligand and measuring the fluorescence intensity each time.

After importing the data into Sigmaplot I plotted this fluorescence intensity against the concentration of DNA to obtain a hyperbola. I performed a dynamic curve fit using the inbuilt one site saturation model under Ligand Binding using the equation:

f = Bmax*abs(x)/(Kd + abs(x))

The results are as follows:

R            Rsqr     Adj Rsqr   Standard Error of Estimate

0.9886 0.9773      0.9748               651.4329

            Coefficient   Std. Error    t              P

Bmax 15353.7677 524.2134   29.2892

The most important number is the value of the coefficient

Kd (dissociation constant of the complex) = 2.48 (the units are in the same concentration units as the titrant)

The standard error (std. error) = 0.44 (the units are the same as the Kd) is a measurement of how precisely the Kd was determined by the data. Since it is small compared to the Kd, it shows that the value of Kd was determined with a fairly high degree of precision. You should express the result of the measurement as Kd = 2.5 +/- 0.4.

Bmax is just the maximal signal that would be obtained once enough titrant has been added to bind up all of the fluorescent ligand. The units are relative fluorescence units (RFU).

Mr. Ravichandran,

Please pay attention to:

[Ref. 1.] Org. Biomol. Chem., 2010, 8, 4949-4959, Effect of substituents of alloxazine derivatives on the selectivity and affinity for adenine in AP-site-containing DNA duplexes, B. Rajendar, A. Rajendran, Z. Ye, E. Kanai, Y. Sato, S. Nishizawa, M. Sikorski, N. Teramae.