Analysis of Binding Interaction between the Natural Apocarotenoid Bixin and Human Serum Albumin by Circular Dichroism and Fluorescence Spectroscopy

Abstract

Bixin is an important, pharmacologically active dietary cis-carotenoid, but its interaction with potential macromolecular targets is completely unexplored. This work was aimed to study the binding of bixin to human serum albumin (HSA), the most abundant protein in blood plasma. Circular dichroism (CD) spectroscopy in combination with UV/VIS absorption spectroscopy and fluorescence quenching techniques were applied. Appearance of induced CD bands in the UV- and VIS-absorption spectral regions indicated the formation of non-covalent carotenoid-albumin complexes. Shape and spectral position of the extrinsic Cotton effects suggested the binding of a single bixin molecule to HSA in chiral conformation. Scatchard and non-linear regression analyses of CD titration data resulted in similar values for the association constant (Ka = 6.6 and 4.6x10(5) M(-1), resp.) and for the number of binding sites (n = 1). The binding interaction was independently confirmed by fluorescence-quenching experiment from which the binding parameters were also calculated. CD Displacement measurements performed with marker ligands established that the main drug binding sites of HSA are not involved in binding of bixin. Palmitic acid decreased the amplitude of the induced CD bands suggesting a common albumin binding site for bixin and long-chain fatty acids. The above data indicate that HSA plays a significant role in the plasma transportation of bixin and related dietary carboxylic acid carotenoids.

Multispectroscopic, virtual and in vivo insights into the photoaging defense mediated by the natural food colorant bixin

“…36 The maximum emission intensity of collagenase (λ em = 334 nm), elastase (λ em = 333 nm), and hyaluronidase (λ em = 337 nm) decreased gradually on interaction with bixin. These findings are in line with earlier reports on bixin forming caroteno-protein complexes with proteins such as HSA 33 and β-lactoglobulin, 26 resulting in fluorescence quenching of these proteins. HSA and β-lactoglobulin are both transporter proteins and a high binding affinity of bixin with these proteins would assist in plasma transportation of bixin; however, the critical gap of probable off-target binding toxicity of bixin needs to be evaluated and bridged by integration of virtual, in vitro, and in vivo advanced screening, prediction, and identification strategies 37 The Stern-Volmer quenching constants (K SV ) were derived from eqn (2) and linear regression of F 0 /F plots against bixin (Fig.…”

“…[30][31][32] Earlier, bixin has been reported to form aggregates in the presence of Ringer buffer due to low aqueous solubility resulting in hypochromism and a blue shift. 33…”