| With the rapid development of animal husbandry, veterinary medicine and the relative additive play an important role in the breeding industry, while the problems they bring are emerging. Clenbuterol hydrochloride is a typical veterinary drug additive. It can promote the decomposition and inhibit the deposition of fat, respectively. In other words, it has a significant nutritional redistribution effect which can improve carcass lean meat. Since CLB can bring huge economic benefits, it has been applied to aquaculture. With its side effects, CLB is prohibited as a veterinary drug now.Proteins and DNA are main component of biological organisms. After entering organisms, the environmental pollutants usually show their toxicity by interacting with the biological macromolecules and affecting the structure as well as function. Therefore, to explore the toxic effects and mechanism of pollutants at molecular level is helpful to perfect the toxicity evaluation system of the pollutants. It is also useful to provide reference and technical support for formulating reasonable standard to protect human body health.In this paper, we studied the toxicity mechanism of clenbuterol hydrochloride (CLB) targeted to bovine serum albumin (BSA), bovine trypsin and calf thymus DNA (ctDNA) at molecule level by multi-spectroscopy methods and molecular modeling. The dissertation consists of the following three parts.In the first part, the interaction mechanism between CLB and BSA was analyzed using fluorescence spectroscopy, ultraviolet-visible absorption spectra, circular dichroism methods and AutoDock software in the simulated physiological conditions. They bound with each other through hydrogen bond and the binding constant is1.124×103L mol-1. The static quenching was caused in process. The BSA skeleton became looser, while the secondary structure and the microenvironment of amino acid residues were changed, which may hinder BSA to exert its normal function, such as transportation and storage. In the second part, we utilized mutil-spectroscopy and molecular docking methods to explore the interaction between trypsin and clenbuterol hydrochloride. Compounds are formed by hydrogen bonding between CLB and trypsin with the binding constant of3.26x104L mol"1. The fluorescence quenching was proved to be static quenching. The trypsin peptides skeleton was looser, leading to the changed secondary and tertiary structure. The microenvironment of the amino acid residues were affected as well as the enzyme activity, which was markedly reduced. This research provides more useful information to better understand the influence of small molecule drugs to the structure and function of enzymes.In the third part, besides traditional spectrum methods (e.g., ultraviolet-visible absorption spectra, the probe fluorescence spectroscopy, circular dichroism), we also employed molecular simulation to obtain a more intuitive result. The groove binding was considered most likely to be the binding mode between them, in consistent with the thermodynamic results, which indicated that the hydrogen bonding and van der Waals force were the main acting force. In addition, the conformation of DNA, namely the double helix structure was affected by the CLB. This research explains the gene toxicity of CLB and provides reference for the investigation in which the neutral red was used as fluorescent probe.The results of our study indicate that CLB can combine with protein and DNA molecules by hydrogen bonds after entering the body. It can affect the structure of macromolecules such as the space conformation and micro environment of amino residues, and even affect the normal function, showing a certain toxicity effect. |
PDF Full Text Request