Praparation Of Water-soluble Chitosan And Study Of Thrombin Immobilized On It

It is widely recognized that thrombin is a high efficient hemostatic and wound-healing accelerator, however, thrombin would not be stable if not used and preserved properly, which can be overcome through immobilization enzyme method. Chitosan which have been used widely as immobilizated carrier of many kinds of enzyme also has the same funtions of hemostasis and wound-healing accelerator as thrombin more all less, while common chitosan could not be used as the carrier of thrombin because of its insolubility in solvent except some special acid solution in which thrombin would lost all of its hemostatic activity. Chitosan with deacetylation degree(D.D.) about 50% were found to become soluble in water and it is called water-soluble chitosan(WSC). It is necessary to make clear whether chitosan with other D.D. value can be used to prepare WSC except chitosan with D.D. of 90% now used only, and it is also important to study the condition and effect of immobilization of thrombin by WSC, which is the basic study of a new kind of thrombin-hemostatic.Being treated with acetic anhydride under homogeneous conditions, Chitosan(D.D.> 80%)can be used to prepare WSC by controlled partial N-acetylation method, during which the key factor is controlling the mol proportion between acetic anhydride and free amino of chitosan with 1:1 (acetic anhydride method) or 2:1 (acetic anhydride-pyrdeine method). It is also suggested mat chitosan (D.D.<70%) couldn't be used to prepare WSC.The aim is to immobilize thrombin with WSC through two methods ( physical adsorption and chemical covalent binding). Activity recycle ratio of immobilized thrombin prepared by physical adsorption method is 81%, higher than that by chemical covalent binding method (56%). In thermal aged experiment, the half life of immobilized thrombin prepared by physical adsorption method is 14 times(normal temperature) , 42 times(40℃) and 12 times(60℃) than that of free thrombin. Among the productions prepared by chemical covalent binding method, these values of the best result are 12 times(normal temperature), 216 times(60℃) ,more over the activity of immobilized thrombin doesn't decrease after aging for 31d in the condition of 40℃. The optimum temperature for immobilized thrombin prepared by these two methods is about 30℃, higher than that of free thrombin(<2 5 ℃)...