Biophysical influence of isocarbophos on bovine serum albumin: Spectroscopic probing

Isocarbophos (ICP) is a phosphorous pesticide with high toxicity. It has been detected in several kinds of food and therefore can enter human body. In this paper, spectroscopic approaches including three-dimensional fluorescence (3D-FL) spectroscopy, UV–visible absorption spectroscopy and circular dichroism (CD) spectroscopy were employed to explore the binding of ICP to bovine serum albumin (BSA) at simulated physiological conditions. It was found that the fluorescence quenching of BSA was caused by the formation of ICP-BSA complex at ground state and belonged to static quenching mechanism. The binding constants, the number of binding sites, enthalpy change (ΔHθ), Gibbs free energy change (ΔGθ) and entropy change (ΔSθ) were calculated at four different temperatures according to Scatchard model and thermodynamic equations. To identify the binding location, fluorescence probe techniques were used. The results showed that warfarin, an acknowledged site marker for BSA, could be partially replaced by ICP when ICP was added to warfarin–BSA systems, which demonstrated that ICP primarily bound on Sudlow's site I in domain IIA of BSA molecule. The distance r (3.06 nm) between donor (Trp-212) and acceptor (ICP) was obtained based on Förster's non-radiation fluorescence resonance energy transfer (FRET) theory. Furthermore, the CD spectral results indicated that the secondary structure of BSA was changed in presence of ICP. The study is helpful to evaluating the toxicology of ICP and understanding its effects on the function of protein during the blood transportation process.

Warfarin which binds specifically to site I has more powerful ability to quench the luminescence of BSA than ICP at equimolar concentration. When ICP was added to BSA–warfarin system, an enhancement of the relative fluorescence intensity could be detected, which suggested that ICP had competed with warfarin for the site I in the subdomain IIA of BSA and warfarin had been partially replaced by ICP.Figure optionsDownload as PowerPoint slideHighlights
► ICP binds with BSA through hydrogen bonds together with hydrophobic interactions.
► The order of the binding constants Kb is 108 L mol−1.
► The binding process is with low reversibility around body temperature.
► The changes of the elaborate second-order structures contents in BSA are calculated.
► ICP binds primarily on Sudlow's site I in domain IIA of BSA molecule.