Azo compounds are the largest chemical class of agents frequently used as colorants in a variety of consumer goods and farm produce; therefore, they may become a hazard to public health, because numerous azo compounds and their metabolites are proven to be carcinogens and mutagens. Herein several qualitative and quantitative analytical techniques, including steady state and time-resolved fluorescence, circular dichroism (CD), computer-aided molecular docking as well as molecular dynamics simulation, were employed to ascertain the molecular recognition between the principal vehicle of ligands in human plasma, albumin and a model azo compound, flavazin. The results show that the albumin spatial structure was changed in the presence of flavazin with a decrease of α-helix suggesting partial protein destabilization/self-regulation, as derived from steady state fluorescence, far-UV CD and detailed analyses of three-dimensional fluorescence spectra. Time-resolved fluorescence further evinced that the recognition mechanism is related to albumin-flavazin adduct formation with an association intensity of 10(4) M(-1), and the driving forces were found to be chiefly π-π interactions, hydrophobic interactions and hydrogen bonds. The specific binding domain of flavazin in protein was defined from molecular docking; subdomain IIA (Sudlow's site I) was found to retain high affinity for the ligand flavazin. This finding corroborates the results of competitive ligand displacement experiments, a hydrophobic 8-anilino-1-naphthalenesulfonic acid probe study and protein denaturation results, placing flavazin at the warfarin-azapropazone site. Based on molecular dynamics simulation, it can be said with certainty that the results of molecular docking are credible, and the key amino acid residues participating in the molecular recognition of flavazin by protein are clearly Trp-214, Arg-222 and Lys-436. The outcomes presented here will help to further comprehend the molecular recognition of azo compounds by protein and the possible toxicological profiles of other compounds that have configurations analogous to azo chemicals.

Malachite green is an organic compound that can be widely used as a dyestuff for various materials; it has also emerged as a controversial agent in aquaculture. Since malachite green is proven to be carcinogenic and mutagenic, it may become a hazard to public health. For this reason, it is urgently required to analyze this controversial dye in more detail. In our current research, the interaction between malachite green and hemoglobin under physiological conditions was investigated by the methods of molecular modeling, fluorescence spectroscopy, circular dichroism (CD) as well as hydrophobic ANS displacement experiments. From the molecular docking, the central cavity of hemoglobin was assigned to possess high-affinity for malachite green, this result was corroborated by time-resolved fluorescence and hydrophobic ANS probe results. The recognition mechanism was found to be of static type, or rather the hemoglobin-malachite green complex formation occurred via noncovalent interactions such as π-π interactions, hydrogen bonds and hydrophobic interactions with an association constant of 10(4) M(-1). Moreover, the results also show that the spatial structure of the biopolymer was changed in the presence of malachite green with a decrease of the α-helix and increase of the β-sheet, turn and random coil suggesting protein damage, as derived from far-UV CD and three-dimensional fluorescence. Results of this work will help to further comprehend the molecular recognition of malachite green by the receptor protein and the possible toxicological profiles of other compounds, which are the metabolites and ramifications of malachite green.

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.

Triterpenoids were thought to be biologically ineffective for a very long time, but aggregating proof on their widely ranging pharmacological activities paired with a dubious toxicity portrait has motivated regenerated attraction for human health and disease. In the current contribution, our central goal was to integratively dissect the biointeraction of two typical triterpenoids, ursolic acid and oleanolic acid, by the most fundamental macromolecule bovine serum albumin (BSA) by employing molecular modeling, steady state and time-resolved fluorescence, and circular dichroism spectra at the molecular scale. Based on molecular modeling, subdomain IIA, which matches Sudlow's site I, was allocated to retain high affinity for triterpenoids, but the affinity of ursolic acid with subdomain IIA is somewhat inferior compared to that of oleanolic acid, probably because the affinity differentiation arises from the different positions of the methyl group on the E-ring in the two triterpenoids. This sustains the site-specific ligands, and hydrophobic 8-anilino-1-naphthalenesulfonic acid probe results in arranging the triterpenoids at the warfarin-azapropazone site. The data of steady state and time-resolved fluorescence indicated that the recognition of triterpenoids by BSA produced quenching by a static type, in other words, the ground state BSA-triterpenoid complex formation with the affinities of 1.507/1.734, 1.042/1.186, and 0.8395/0.9863 × 10(4) M(-1) at 298, 304, and 310 K for ursolic acid/oleanolic acid, respectively. Thermodynamic analyses show that the basic forces acting between BSA and triterpenoids are hydrogen bonds, van der Waals forces, and hydrophobic interactions; this occurrence provoked the alterations of the BSA spatial structure with a noticeable decline of α-helix evoking perturbation of the protein, as stemmed from circular dichroism, synchronous fluorescence, and three-dimensional fluorescence measurements. We anticipate that the complexation of plant triterpenoids with protein delineated here may be exploited as a biologically relevant model for evaluating the physiologically applicable noncovalent complexes in in vivo examination of triterpenoid properties such as accumulation, bioavailability, and distribution.

Flubendiamide, a ryanoid class insecticide, is widely used in agriculture. Several insecticides have been reported to promote adipogenesis. However, the potential influence of flubendiamide on adipogenesis is largely unknown. The current study was therefore to determine the effects of flubendiamide on adipogenesis utilizing the 3T3-L1 adipocytes model. Flubendiamide treatment not only enhanced triglyceride content in 3T3-L1 adipocytes, but also increased the expression of cytosine-cytosine-adenosine-adenosine-thymidine (CCAAT)/enhancer-binding protein α and peroxisome proliferator-activated receptor gamma-γ, two important regulators of adipocyte differentiation. Moreover, the expression of the most important regulator of lipogenesis, acetyl coenzyme A carboxylase, was also increased after flubendiamide treatment. Further study revealed that 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) or A769662, two Adenosine 5′-monophosphate (AMP)-activated protein kinase α activators, subverted effects of flubendiamide on enhanced adipogenesis. Together, these results suggest that flubendiamide promotes adipogenesis via an AMPKα-mediated pathway.