Investigation of hexacyanoferrate(II)/(III) charge-dependent interactions with bovine and human serum albumins

Abstract

In the present paper, the binding interactions of highly negative-charged ions, namely hexacyanoferrates(II/III), i.e. [Fe(CN)₆]^4- and [Fe(CN)₆]^3- with bovine and human serum albumins (BSA and HSA, respectively) have been studied for the first time in an aqueous solution (10 mM cacodylate buffer of pH 7.0) using steady-state fluorescence spectroscopy, isothermal titration calorimetry, and CD spectroscopy supported by molecular dynamics-based computational approaches. The Stern-Volmer equation as well as its modifications suggested that hexacyanoferrates(II/III) effectively quenched the intrinsic fluorescence of the albumins through a static mechanism. The proteins under study possess only one binding site on the surface capable of binding one mole of hexacyanoferrates(II/III) ions per one mole of albumin (HSA or BSA). The formation of albumin complexes is an enthalpy-driven process (|ΔH_ITC| > |TΔS_ITC|). The strength of the interactions depends mainly on the type of albumin, and changes as follows: BSA-K₃[Fe(CN)₆] ∼ BSA-K₄[Fe(CN)₆] > HSA-K₃[Fe(CN)₆] ∼ HSA-K₄[Fe(CN)₆]. Finally, potential binding sites of bovine and human serum albumins have been investigated and discussed based on a competitive fluorescence displacement assay (with warfarin and ibuprofen as site markers) and molecular dynamics simulations.

Keywords: Binding properties; Bovine and human serum albumins; CD spectroscopy; Fluorescence quenching; Isothermal titration calorimetry; Molecular dynamics simulations; Potassium hexacyanoferrates(II/III); Steady-state fluorescence spectroscopy.