Study on the Luminescence Behavior of Model Proteins with Active Components of Chinese Medicine and Its Application
|Keywords||Model protein Active components of traditional Chinese medicine Chemiluminescence Flow injection analysis|
The binding of proteins with drugs has a great influence upon the distribution, patterns of metabolism and excretion of the drugs. And it also has influence on the curative effect of the drugs. So the study on the binding of drugs with proteins is very important to explain the pharmacology activities and pharmacokinetics of the drugs.Rhubarb is one of four Chinese medicines which are often used in clinic. The active components of rhubarb are anthraquiones, including emodin, chrysophanol, rhein, aloe-emodin and physcion. Rutin, kaempferol and hesperetin, which belong to flavonoids, are often used in clinic. Rudin has been produced in oral dosage in China.The binding of myoglobin with anthraquiones and flavonoids was studied in this dissertation by using luminol as chemiluminescence probe based on FI-CL method. The following major innovative works was carried out in this dissertation.1. The interaction of BSA, Mb, lysozyme and CAT with luminol has been investigated fully by taking luminol as the phosphor. The new mathematical model was established that can be used to study the binding of proteins with small molecule compounds by determining the fluorescence of the small molecule compound. And the binding parameters such as the binding constant, the number of binding sites were calculated by this model.2. The new FI-CL mathematical mode has been established which can be used to study the binding of Mb with drugs by using luminol as chemiluminescence probe. The binding constant and the number of binding sites were calculated for the interaction of anthraquiones and flavonoids with myoglobin by this mode. The results showed it is sample and sensitive to study the interaction between proteins and drugs by using this mode. And some valuable reference parameters can be obtained (indluding bingding constant, the number of binding sites, binding mode and the changes of thermodynamic parameters). It provided the new method and technology for explaining the binding mechanism between proteins and drugs.The dissertation cinsisted of the following main content:1. The structure and biological functions of proteins and research methods for the interaction between proteins and small molecules were described. The pharmacology activities of Rhubarb, anthraquiones and flavonoids were reviewed.180 references were cited. 2. According to the Stern-Volmer equation, it was induced that the quenching process of model proteins-luminol was the static quenching mode to form the nonradiative ground-state complex. Based on this method, the new mathematical model was established that can be used to study the binding of small molecule compounds with proteins by determining the fluorescence of the small molecule compound. The mathematical equation is: The binding constants and number of binding sites were calculated for model proteins-luminol by this model. The possible binding sites were discussed according to the structure of model proteins.3. The influence of model proteins (BSA, Mb, lysozyme and CAT) on the chemiluminescence of luminol was studied by FI-CL method. The results showed model proteins enhanced the chemiluminescence of luminol, and Tmax was changed smaller. The increase in the chemiluminescence intensity was proportional to the concentration of model proteins. The possible mechanism of luminol-model protein systems was provided.4. The influence of emodin, chrysopanol, rhein, aloe-emodin and physcion on chemiluminescence of luminol-Mb system has been investigated. Based on the inhibitory effect, the possible mechanism was discussed and the new FI-CL mathematical mode was established. The mathematical equation is The binding parameters such as the binding constant, the number of binding sites were calculated by it for Mb-anthraquiones at 298K and 303K. The values of the binding constants were at 103～107 level. The binding ability of the studied anthraquiones followed the pattern: aloe-emodin> chrysophanol>emodin>physcion>rhein. The changes of thermodynamic parameters were calculated and they showed Mb bound with anthraquiones by hydrophobic effect. The relationship between binding constants of them with their structure and pharmacology activities was discussed.5. The influence of rudin, kaempferol and hesperetin on chemiluminescence of luminol-Mb system has been investigated. Based on the inhibitory effect of them, the possible mechanism was provided. The binding parameters such as the binding constant, the number of binding sites were calculated by the new FI-CL mathematical mode at 298K and 303K. The values of the binding constants were at 104～105level. The binding ability of the studied flavonoids followed the pattern:kaempferol> rudin> hesperetin. The changes of thermodynamic parameters were calculated and they showed Mb bound with flavonoids by hydrophobic effect. The relationship between binding constants of them with their structure was discussed.6. The inhibitory effect on luminol-Mb chemiluminescence of clindamycin, loratadine, melamine and gentiopicroside was studied. The binding constant and the number of binding sites were calculated by the new FI-CL mathematical mode for the interaction of Mb with them. Because clindamycin, loratadine and melamine reacted with Mb firstly, the calculating results were satisfied.