Binding Patterns and Structure–Affinity Relationships of Food Azo Dyes with Lysozyme: A Multitechnique Approach

Abstract

Food dyes serve to beguile consumers: they are often used to imitate the presence of healthful, colorful food produce such as fruits and vegetables. But considering the hurtful impact of these chemicals on the human body, it is time to thoroughly uncover the toxicity of these food dyes at the molecular level. In the present contribution, we have examined the molecular reactions of protein lysozyme with model food azo compound Color Index (C.I.) Acid Red 2 and its analogues C.I. Acid Orange 52, Solvent Yellow 2, and the core structure of azobenzene using a combination of biophysical methods at physiological conditions. Fluorescence, circular dichroism (CD), time-resolved fluorescence, UV-vis absorption as well as computer-aided molecular modeling were used to analyze food dye affinity, binding mode, energy transfer, and the effects of food dye complexation on lysozyme stability and conformation. Fluorescence emission spectra indicate complex formation at 10(-5) M dye concentration, and this corroborates time-resolved fluorescence results showing the diminution in the tryptophan (Trp) fluorescence mainly via a static type (KSV = 1.505 × 10(4) M(-1)) and Förster energy transfer. Structural analysis displayed the participation of several amino acid residues in food dye protein adducts, with hydrogen bonds, π-π and cation-π interactions, but the conformation of lysozyme was unchanged in the process, as derived from fluorescence emission, far-UV CD, and synchronous fluorescence spectra. The overall affinity of food dye is 10(4) M(-1) and there exists only one kind of binding domain in protein for food dye. These data are consistent with hydrophobic probe 8-anilino-1-naphthalenesulfonic acid (ANS) displacement, and molecular modeling manifesting the food dye binding patch was near to Trp-62 and Trp-63 residues of lysozyme. On the basis of the computational analyses, we determine that the type of substituent on the azobenzene structure has a powerful influence on the toxicity of food dyes. Results from this work testify that model protein, though an indirect method, provides a more comprehensive profile of the essence of toxicity evaluation of food dyes.

Elucidation of Binding Mechanism of Photodynamic Therapeutic Agent Toluidine Blue O with Chicken Egg White Lysozyme by Spectroscopic and Molecular Dynamics Studies

“…reported an identical behavior for the binding of lysozyme with C.I. Acid Red 2 dye . In the present case, we believe that the monitored changes in the emission intensity are indicative of TBO complexation with lysozyme.…”

“…The exposure of Trp residues of lysozyme to the solvent polarity upon addition of TBO resulted in the quenching of lysozyme. As a result, the noticed drop in the emission intensity may be due to the lysozyme–TBO complexation and the binding site of lysozyme for TBO is located adjacent to the Trp residues of lysozyme . Further, the emission spectrum of TBO upon the addition of lysozyme is also presented in Fig.…”

Chelerythrine–lysozyme interaction: spectroscopic studies, thermodynamics and molecular modeling exploration

“…61 Consequently, we have not tried to designate the individual components; conversely, the average fluorescence lifetime has been exploited for qualitative analysis. From the time-resolved fluorescence decay profiles of Lys in the absence and presence of chelerythrine (not shown) the fluorescence lifetime values and their amplitudes were calculated.…”

“…From the time-resolved fluorescence decay profiles of Lys in the absence and presence of chelerythrine (not shown) the fluorescence lifetime values and their amplitudes were calculated. [61][62][63] Steady-state fluorescence anisotropy study Steady state anisotropy data may provide valuable information on the degree of motional restriction of the fluorophore and is used as a probe to assess the extent of the flexibility or tumbling motion of small molecules after binding. After adding the maximum concentration of the chelerythrine iminium and alkanolamine forms (100 mM) to Lyz, the observed average fluorescence lifetime values were t = 1.81 ns and 2.07 ns, respectively.…”

“…74 Considering this, we compared the chelerythrine binding region to that of the amyloidogenic aggregation prone region. Most of them make either a direct or indirect contribution to fibril formation, coming from K-peptide (54)(55)(56)(57)(58)(59)(60)(61)(62) 15 or amyloid state protelytically resistant (32-108) 79 regions, respectively. Previously, 16 amino acids have been reported to be essential for aggregation activity.…”