College of Food Science & Engineering, Northwest A&F University , Yangling 712100, China.
J Agric Food Chem. 2013 Dec 18;61(50):12415-28. doi: 10.1021/jf4039327. Epub 2013 Dec 9.
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.
它们通常被用来模仿水果和蔬菜等有益健康、色彩鲜艳的食品。但是，考虑到这些化学物质对人体的有害影响，现在是时候从分子水平上彻底揭示这些食品染料的毒性了。在本研究中，我们使用生理条件下的多种生物物理方法，研究了蛋白质溶菌酶与模型食品偶氮化合物食品添加剂索引号（C.I.）酸性红 2 及其类似物 C.I.酸性橙 52、溶剂黄 2 和偶氮苯的核心结构之间的分子反应。荧光、圆二色性（CD）、时间分辨荧光、紫外-可见吸收以及计算机辅助分子建模被用于分析食品染料的亲和性、结合模式、能量转移以及食品染料络合对溶菌酶稳定性和构象的影响。荧光发射光谱表明，在 10(-5) M 染料浓度下形成复合物，这与时间分辨荧光结果相符，该结果表明色氨酸（Trp）荧光的减弱主要通过静态（KSV = 1.505 × 10(4) M(-1))和福斯特能量转移。结构分析表明，几个氨基酸残基参与了食品染料-蛋白质加合物的形成，存在氢键、π-π和阳离子-π相互作用，但从荧光发射、远紫外 CD 和同步荧光光谱来看，溶菌酶的构象在该过程中没有改变。整体而言，食品染料的亲和力为 10(4) M(-1)，且蛋白质中只有一种结合域用于结合食品染料。这些数据与疏水性探针 8-苯胺-1-萘磺酸（ANS）置换一致，分子建模也表明，食品染料的结合区域靠近溶菌酶的色氨酸 62 和色氨酸 63 残基。基于计算分析，我们确定偶氮苯结构上取代基的类型对食品染料毒性有很大影响。这项工作的结果证明，模型蛋白虽然是一种间接方法，但为食品染料毒性评估提供了更全面的本质特征。
