Biotoxicity Of Azo Dyes And Their Binding With Biomacromolecules

The toxicity of dyes, especially azo dyes, has been highly paid attention to by all governments around the world. How to rapidly evaluate the toxicity effect of azo dyes to the ecological environment in order to minimize its negative effects has become a pressing research issue. Dye molecules can be absorbed through various channels by the human body, causing certain biological effects. Especially their binding with key biological macromolecules in the target areas results in abnormal structure and function of biological macromolecules, thus causing poisonous effects. In this dissertation the toxicity of three azo dyes was studied on two levels. First, their acute toxicity was evaluated by luminous bacteria method and the mechanism of toxicity was analyzed by molecular docking technology. To predict potential dangers to human beings, we conducted research on the molecular mechanism of three kinds of azo dyes binding to human serum albumin, and on the possible mode of binding of the dyes with DNA. The main contents and results are following:1. The acute toxicity of three azo dyes (C.I. Acid red 73, C.I. Acid blue 113 and C.I. Reactive red 24) was evaluated by bioluminescence tests. Raman shift of biological macromolecules in luminous bacteria before and after exposing dyes was measured. The results were shown that all the three kinds of azo dyes contain certain level of toxicity within the range of experiment concentration. The descending order of their toxicity is:C.I. Acid red 73> C.I. Acid blue 113> C.I. Reactive red 24. Among them, C.I. Acid red 73 has the highest toxicity. Luminescence inhibition ratio of C.I. Acid red 73 (100 mg/L, exposing 15 min) is 26.9%, whose toxicity equals to HgCl2 at the concentration level of 0.061 mg/L, and the longer the time the higher the toxicity level. Raman spectrum of the luminous bacteria also shows that the three azo dyes either combined with their biological macromolecules or changed the micro-environment of macromolecules, resulting in various changes in the strength and location of Raman peaks, which further influences the normal physiological metabolism processes of luminous bacteria reflected by their reduced luminescence strength. 2. The best binding site of flavin mononucleotide (FMNH2) and bacterial luciferase was studied applying molecular docking technology, and completed molecule docking research on FMNH2 and three areas (αsubunit,βsubunit and the interface between the two subunits) of the bacterial luciferase. Based on the lowest binding energy of FMNH2 and the three areas, two active sites were identified, one locates at a subunit, designated as active site-Ⅰ, which is consistent with the abundance of experimental results reported previously; the other locates at the interface between two subunits, designated as active site-Ⅱ. The binding energy of FMNH2 on the two sites is-32.4 KJ mol-1 (active site-Ⅱ) and -31.2 KJ mol-1 (active site-Ⅰ), indicating a similar affinity on the two sites. The introduction of active site-Ⅱhelps to understand why theα/βheterodimer has higher catalytic activity than the individualαandβsubunits, and provide theoretical basis for the study of how chemical contaminants affect luminescence strength of the bacteria as well as the studies on gene mutations and molecular dynamics simulation.3. Molecular docking technology was applied to complete the docking simulation of four long chain aliphatic aldehydes and the three azo dyes on the two active sites of the bacterial luciferase. The lowest binding energy of each substance on these two sites was calculated. The molecular mechanism of inhibiting the luminescence strength of bacteria of three dyes was analyzed through comparing the binding energy. As is shown by the molecule docking experiment, comparing to the FMNH2, C.I. Acid red 73 is less competitive on the two sites, but it is able to inhibit the binding of and the long chain aliphatic aldehyde on the two active sites, thus possesses great potential in inhibiting luminescence. Whereas both C.I. Acid blue113 and C.I. Reactive red 24 have high affinity on active site-Ⅰand are able to prevent the binding of enzymes and the two objects on active site-Ⅰ, but have low affinity on active site-Ⅱshown by their positive value of binding energy, thus cannot inhibit their binding on active site-Ⅱand luminescence of bacteria. This is consistent with the results of luminescence experiment.4. The molecular mechanism of the three dyes binding to human serum albumin (HSA) was investigated by fluorescence, UV-visible, far-UV CD spectroscopy and molecule docking technology. Theories and experiments have revealed high affinity ability between the three dyes and the protein to spontaneously form coordination complexes. The hydrophobic interaction is the main pattern of driving force, and the hydrogen bond and static electricity effect also play an important role in forming stable dye-protein compounds. Besides, a small change in the secondary structure of the protein was also induced by the combination of dyes and the protein.As the results of molecule docking shows, the most possible binding site of the two acidic dyes C.I. Acid red 73 and C.I. Acid blue113 and the protein locates within the IB sub-domain of the protein. The average distances between the donor (Trp214) and the acceptor (dyes) are 3.06 and 2.82 nm respectively, which are very similar to the results from the experiment:3.28 nm (C.I. Acid red 73) and 2.78 (C.I. Acid blue 113). The main binding area of the C.I. Reactive red 24 is within the IIA sub-domain of the protein. The average distance between Trp214 and C.I Reactive red 24 is 1.18 nm, while the result obtained through Forster energy transfer theory is 3.11 nm, indicating certain level of error between the two. The reasons for this could be:(1) the molecules of C.I. Reactive red 24 are rich in active groups, including negatively charged three-SO3, two-Cl and a-OH as well as positively charged di-amine and a triazine ring. These active particles facilitate the binding of molecules to amino acid in the protein, either alkalescent or acidic, instead of binding to the four sites under this study; (2) IIA sub-domain is the best binding site for C.I. Reactive red 24, but there are also some molecules binding in other sub-domains. The distance obtained through Forster energy transfer theory should be the average of the distances between all the binding sites of C.I. Reactive red 24 and Trp214; further research needs to be done to uncover other reasons.According to the results, among the three dyes, C.I. Acid red 73 has the lowest binding free energy△G (the results from the experiment) and highest binding constant Kb, which is consistent with the fact that it has the highest toxicity (shown by bioluminescence tests). The binding of dyes with protein may affect the storage and transportation of vital substances in the organism by forming a competition, which consequently disturbs normal physiologic function leading to the occurrence of toxicity. Therefore, through investigating the binding of dye with protein, it is possible to predict biological toxic by calculating their binding energy and binding sites.5. Molecular docking techniques were applied to describe the most probable mode of nine DNA fragments binding as well as the sequence selectivity of the three dyes. As shown by the molecule docking experiment, the minor groove binding is the main pattern of the three dyes binding to normal DNA fragments (without intercalation gaps). The minor groove binding is the most preferable binding model of some dyes although DNA targets present intercalation gap. By analyzing the binding model, only the naphthalenedisulfonic acid moiety of the C.I. Acid red 73 selectively binds to the CG-rich region of DNA minor groove, while other dyes are not obviously selective to sequences of base pairs. Research shows that the affinity of the three dyes to DNA relates to the number of aromatic groups bound to the minor groove, and the more the aromatic rings the greater the affinity. Comparing to other two dyes, due to more aromatic rings bound to the minor groove to form a biggerπ-π-stack in C.I. Acid blue 113, apart from one dodecamer, C.I. Acid blue 113 has the highest binding energy to other eight DNA fragments. Further research needs to be done to decide whether this indicates greater genetic toxic of the dye.The major groove of DNA is the place of combining proteins where dye molecules with some conformation combined in this study. If the dye molecules form a competition with proteins, thus influencing the recognition of proteins, the normal physiological function of organism may be disturbed, leading to the production of toxicity.