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Graphical abstract Highlights • The interaction between sitafloxacin and human serum albumin has been firstly investigated. • Quenching mechanism, binding constants, thermodynamic parameters, and binding modes were discussed and mutually confirmed. • Molecular dynamics simulations were performed to further analyze the HSA-sitafloxacin complex systems. • The secondary structure of the protein belonging to each amino acid residue in the HSA were analyzed. Abstract Sitafloxacin (STFX) is a new generation of broad-spectrum oral fluoroquinolones. STFX has significantly enhanced antibacterial activity than most similar drugs. Clinically, this drug is mainly used to treat respiratory and urinary tract infections and other serious bacterial infections. In this study, the interaction between sitafloxacin and human serum albumin (HSA) was investigated by spectroscopic methods and molecular simulations. Fluorescence quenching experiments showed that the interaction mechanism between STFX and HSA was static quenching, which was confirmed by time-resolved fluorescence. Thermodynamic parameters and docking results indicated that hydrophobic and electrostatic forces played a key role in this mechanism. Probe experiments and molecular docking results indicated that the major binding of STFX was at site I. 3D fluorescence showed that the insertion of STFX had minimal impact on the microenvironment. Analysis of the protein secondary structure showed that the insertion of STFX had little effect on the secondary structure of the protein. Hydrophobicity experiments showed the slight decrease in the overall hydrophobicity index of the system. Molecular dynamics simulations further validated the stability of the HSA-STFX complex. This study are useful for further drug development, in vivo toxicity studies, and can provide guidance for the clinical application of STFX to study its pharmacokinetic properties. [ABSTRACT FROM AUTHOR]

Development of powerful intramolecular charge transfer (ICT) fluorescent probes that can recognize, detect, and visualize human serum albumin (HSA) is important in the fields of biology and biomedicine. Three ICT probes, namely, butyl gallate (BG), isobutyl gallate (IBG), and isoamyl gallate (IAG), which have simple structures, were selected and used to detect HSA. The probes selectively recognized HSA in aqueous solutions by forming stable complexes with the protein. All of the probes exhibited different changes in fluorescence, and HSA underwent significant changes in fluorescence intensity and decay time. We hypothesized that the probes formed electromagnetic coupling with the whole HSA. Not all biomolecules can offer favorable environment for electromagnetic coupling due to their complex structures. The probes shared the same main binding site (located in Sudlow site II) in HSA. The probes and warfarin fluorescence intensity evidently increased in different hydrophobic cavities of HSA. This finding indicated the lack of limit to the location of the probes. A more suitable electromagnetic environment in protein made the ICT probes all had better response ability in HSA than in BSA. Y-type alkyl chains in IBG and IAG were beneficial for their responsiveness. The binding and response abilities of a specific hydrophobic cavity of a protein should be comprehensively considered when designing a novel fluorescent probe. Image 1 [ABSTRACT FROM AUTHOR]

[Display omitted] • The hydrophilic group of T6 decreased the binding constant compared with T1. • HSA in the T6 system undergo a more significant conformational transition. • T1 and T6 quenched the endogenous fluorescence of HSA via static mechanism. • Both systems were spontaneous reactions driven by hydrophobic interaction. Introduction of hydrophilic groups can improve the solubility of leading drugs but inevitably affect their interaction with proteins. This study selected sirtuin inhibitors Tenovin-1 (T1) and Tenovin-6 (T6) as drug models to determine differences in binding mode to human serum albumin (HSA). T1 and T6 quenched the endogenous fluorescence of HSA via static quenching mechanism. Introduction of hydrophilic groups greatly reduced the binding constant, i.e., from 1.302 × 104 L mol−1 for the HSA-T6 system to 0.128 × 104 L mol−1 for the HSA-T1 system. HSA-T1 system was mainly driven by electrostatic interactions while that of HSA-T6 system was hydrophobic interaction and both systems were spontaneous reactions. Site marker experiments and molecular docking indicated that both systems mainly bound to the hydrophobic site I of HSA. Molecular dynamics (MD) simulation analysis further revealed that Tyr148, Tyr150 and Arg257 residues played a key role in this recognition process for both systems. In particular, T6 maintained additional several hydrogen bonds with the surrounding residues. T1 had almost no effect on the esterase-like activity of HSA, but T6 inhibited the hydrolysis of p-NPA. Furthermore, differential scanning calorimetry (VP-DSC), circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy confirmed that HSA in the T6 system undergone a more significant conformational transition than that in the T1 system. [ABSTRACT FROM AUTHOR]

[Display omitted] • Saturation Transfer Difference–NMR map the interaction based on the ligand. • Molecular recognition pattern and binding epitope of VB12 and HSA are confirmed. • VB12 stretch along the α-helix of HSA in a double-sites binding pattern. • VB12 statically bind to HSA in 104 L/mol sustained by non-covalent forces. • HSA-VB12 is temperature-sensitive and caused obvious protein structure changes. Ligand–receptor molecular recognitionis the basis of biological process. The Saturation Transfer Difference–NMR (STD–NMR) technique has been recently used to gain qualitative and quantitative information about physiological interactions at atomic-resolution. The molecular recognition patterns between Vitamin B12 (VB12) and human serum albumin (HSA) were investigated by STD–NMR supplemented by other spectroscopies and molecular docking. STD–NMR delivered a complete picture that the substituent groups on the tetrapyrrole ring of VB12 interacted with site III of HSA through binding epitope mapping and competitive probe experiments. STD–NMR and fluorescence results proved the moderate binding capability of VB12 and clarified a static, spontaneous, and temperature-sensitive binding mechanism. 3D-fluorencence, FT-IR and circular dichroism spectra showed a compact protein structure by interacting with VB12. Size distribution and surface hydrophobicity showed the surface properties changes of HSA caused by the binding of VB12. Computer simulation confirmed the recognition mode in theory and was compared with experiments. This work is beneficial for understanding the safety and biological action of VB12, and will attract researchers interested in NMR technology. [ABSTRACT FROM AUTHOR]