Fluorescence Spectroscopic Studies to Evaluate Binding Interaction between Hoechst 33258 and Bilirubin

A detailed spectroscopic study (fluorescence, absorption, and lifetime) was conducted to gain insight into the nature of the binding interaction between fluorophore Hoechst33258 (H258) and jaundice marker Bilirubin (Br). The fluorescence emission of the H258 (Ex/Em = 340-502nm) showed a conc. dependent quenching in the presence of Br (1.25 $$ M to 10 $$ M). The Stern-Volmer constant demonstrated an upward curve depicting the occurrence of both static and dynamic quenching with an acquired value of K $$ = 3.1x 10 $$ M $$ and biomolecular quenching rate constant K $$ = 8.6 x 10 $$ M $$ S $$ . The static quenching was evaluated using the sphere of action model and a sphere radius of 0.3nm indicated the presence of a static component in the quenching. The FRET analysis with overlap integral (J) = 1.4x10 $$ M $$ cm $$ nm $$ and Foster Radius(R $$ ) = 26.82 Å with 59% efficiency suggested occurrence of dynamic quenching. Further studies with the time-resolved fluorescence also indicated the presence of dynamic quenching. The lifetime values of H258 reduced from 3.9ns to 0.5ns. Molecular docking studies further support both static and dynamic components in quenching. A non-covalent interaction of H258 with Br in the presence of HSA is predominantly characterized by H-bonding with residues Lys, Asn, Glu, Gln, and Br. The H258 and Br interaction was within the distance of 3.04 Å, which is in coherence with the sphere of action model (0.3nm) and Van-der-Waals along with hydrophobic interactions, which suggested both static and dynamic quenching. Thus, H258 can serve as an efficient fluorophore to monitor binding interactions and can be further exploited as a suitable probe for investigating conformational changes and detection of Br in subsequent studies.