Deciphering the binding site and mechanism of new methylene blue with serum albumins: A multispectroscopic and computational investigation

• Published in: Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy Molecular and biomolecular spectroscopy, 2023-11, Vol.300, p.122900, Article 122900
• Main Authors: Manivel, Perumal, Marimuthu, Parthiban, Ilanchelian, Malaichamy
• Format: Article
• Language: English
• Subjects: Bovine serum albumin; Human serum albumin; Molecular dynamic simulation; New methylene blue; Steady-state emission spectra
• ISSN: 1386-1425
• DOI: 10.1016/j.saa.2023.122900

[Display omitted] •Interaction mechanism of NMB with SAs examined through experimental and Insilico approach.•Emission studies established the static quenching mechanism between SAs and NMB.•Molecular docking analysis revealed the stabilization of NMB inside the binding pocket Subdomain IIA (Site I) of SAs.•Insilico findings were correlates well with the emission results. Herein, the interaction mechanism of new methylene blue (NMB) with human serum albumin (HSA) and bovine serum albumin (BSA) was carefully investigated both experimentally and conceptually, employing experimental and insilico analysis. The steady-state emission spectral studies showed that the emission intensity of HSA and BSA was quenched significantly by NMB. The findings of the Stern–Volmer and double logarithmic plot revealed that the observed emission quenching process was through a static quenching mechanism and the measured binding constant values (Kb) for HSA–NMB and BSA–NMB are 2.766 and 1.187 × 105 dm3 mol−1 respectively. The time-resolved fluorescence lifetime measurement and UV–vis absorption investigation further verify the complex formation between NMB and HSA/BSA. The assessment of thermodynamic parameters disclosed the binding process was spontaneous driven by hydrogen bonds (H-bond) and van der Waals interactions, which contributed a significant role in the complexation. Moreover, the secondary structural conformation and microenvironment of HSA/BSA were modified in the presence of NMB, as evidenced by circular dichroism and synchronous fluorescence data. Molecular docking study predicted a plausible binding mode of NMB inside the binding pocket of HSA and BSA. These results demonstrated that the stabilized NMB is found at the Subdomain IIA (site I) of both the proteins and the results were correlated well with the competitive binding assay. Additionally, the principal components analysis revealed less variation of docked poses for HSA, while, more dispersed docked poses were observed for the BSA model. This also highlights the effects of docking towards a modeled protein (BSA). Molecular dynamic (MD) simulation based binding free energy (ΔGmmgbsa) estimation obtained at 298, 303, 308 and 313 K, were in good agreement with our experimental (ΔGbind) values.