*3.8. Fluorescence Quenching Measurements*

The edG binding studies with heparan sulfate were performed by Jasco spectrofluorometer (FP6200) at 25 ± 1 ◦C in a quartz cuvette with a path length of 1 cm. The stock solution (1 mM) of Heparan sulfate (HS) was prepared in Tris buffer (20 mM) at pH 8.0. The increasing concentration (2 to 50 µM) of heparan sulfate was used to titrate against the fixed protein concentration. The protein excitation was done at 280 nm, and emission spectra were recorded from 300–430 nm with excitation slit at 5 nm and emission slit at 10 nm. The edG showed the emission maxima peak at 344 nm. The final spectra were obtained by subtracting the blank one (heparan sulfate with buffer).

The fluorescence quenching of edG with heparan sulfate was determined to know the different binding parameters such as the Stern–Volmer constant (*KSV*), the binding constant (*K*) and the number of binding sites (*n*).

To determine the Stern–Volmer constant and analyze the quenching data, the Stern– Volmer Equation (2) was used:

$$\frac{F\_0}{F} = 1 + K\_{SV}[\mathbb{C}] \tag{2}$$

where *F<sup>0</sup>* denotes the intensity of protein absence of HS, F denotes the intensity of protein at a specific concentration of HS at 344 nm, [*C*] denotes the different concentrations of HS, and *KSV* is the obtained Stern–Volmer quenching constant.

The bimolecular quenching constant (*Kq*) was calculated using Equation (3) to confirm the quenching mode of the protein–HS complex

$$K\_q = \frac{K\_{SV}}{\tau\_o} \tag{3}$$

where *<sup>τ</sup><sup>0</sup>* is the average integral fluorescence lifetime of tryptophan (2.7 <sup>×</sup> <sup>10</sup>–9 s)

Using Equation (4), the modified Stern–Volmer constant gives a binding constant of the protein–HS complex.

$$\log\left(\frac{F\_0 - F}{F}\right) = \log K + n \tag{4}$$

where *K* denotes the binding constant of protein-HS complex and *n* denotes the number of binding sites.
