| Enzymes, the foundation of all life, promote the metabolic action in vivo. An enzyme inhibitor can regulate the body’s function by inhibiting the activity of its corresponding enzyme. Therefore, it is very important to exploit quick analysis method of enzyme-inhibitor interaction for the development, screening and optimization of small molecular inhibitor drug. Currently, the main research methods include spectroscopy, electrochemical method, nuclear magnetic resonance (NMR), mass spectrometry (MS), and molecular modeling. However, spectroscopy method requires the higher purity of receptor protein, which is particularly unfavorable to the rare protein. Electrochemical method is limited to the electrical activity of the drug molecules at a certain extent. Using NMR and MS can obtain more information, but can not achieve quantitative analysis, what’s more, they are time-consuming, laborious, and require expensive instruments. Molecular modeling becomes more and more important in inhibitor design and screening, but it may have a larger inaccuracy.Aiming at those problems, we established a quick analysis method to study enzyme-inhibitor interaction by using magnetic microspheres (MM). The aim of our study includes three parts:(1) Preparation of the functional MM.(2) Application of the heparin-functioned MM on the purification of thrombin.(3) Application of thrombin-coated MM on the determination of thrombin-inhibitor interation.Firstly, we prepared the amino functioned polystyrene MM (PS-MM) using lawyer-by-lawyer assembly method. Then prepared the heparin-functionalized MM by connecting the carboxy of heparin with amino of the MM using coupling reagents EDC/NHS. The particle size of the heparin-functionalized MM was about1m, monodispersed, and the heparin bond content was5mg/g, with a high biological activity. Ferroferric oxide MM was prepared by a solvothermal reduction method. After modified by amino, prepared the thrombin-coated ferroferric oxide MM by connecting the carboxy of thrombin with amino of MM using coupling reagents EDC/NHS. Thrombin bond content was determination by Bradford method. The effect on the thrombin bond content of concentration of coupling agent was investigated.The specific selectivity of the heparin functionalized MM to the target protein thrombin was investigated using BSA as impurity protein. The purification process of thrombin was optimized. The result of polyacrylamide gel electrophoresis proved that the heparin functionalized MM purification process got a good result to thrombin. Isothermal adsorption experiments of the thrombin-coated MM and argatroban was tested, and the binding constant was calculated by substituting the measured results to the Scatchard model. The thermodynamic parameters were obtained by measuring the binding constant under different temperatures, and thereby deduced the interaction between the enzyme and the drug.The measured binding constant was consistent with the reported result. And this method has been proved that it was simple, fast, intuitive and low cost. The measure method based on MM provided a new approach for the interaction studies between biomolecules and drug. |
