Spectroscopy Studies Of Trypsin After Treatment With Alkaloid

Trypsin is a kind of protease with high speciality, it only catalyses the hydrolyzation of peptide constituted by L-Arg and L-Lys. Being an important digest enzyme trypsin has been widely used in medicine, food industry and organic synthesization and people had done deeply research in this. Now the hot spot of research is the nature inhibitor of trypsin.Alkaloid is a natural heterocyclic compound containing nitrogen, it widely exists in plants, and many of them have medical applications. In this article, we did some research in interaction between three kind of alkaloid and trypsin by spectral analysis on the basis of officinal value.Puerarin, colchicine and camptothecin are chosen for studying the mechanism. Puerarin is isolated from the puerarin, which is widely used in pharmacology. It mainly used in treatment on heart and brain blood vessel disease; colchicines is isolated from corm of Colchicumautumnale and used in curing gout, especially in anti malignant tumor; Camptothecin is a kind of toxic cell alkaloid which is purified from the root and fruit of natural camptotheca acuminate in China. It can be used in treatment of leukemia. The study of interaction between these alkaloid and trypsin can offer some valuable basic theory dates, furthermore , it can illustrate the conformation changes of trypsin in interaction with small molecular.Spectroscopy is developed recently for studying the conformation change of protein in solution. Some structural informations such as the location and the microenvironment of Try in protein, the change of second structure and tertiary structure can be attained from the UV absorption spectra, while Fluorescence can be used in the study of conformation changes ofprotein and the microenvironment of Try and Tip etc.The UV absorption spectra study of trypsin after treatment with these alkaloid showed that the UV absorption peak has changed in two ways: increasing in intensity and blue shift. The difference caused by concentrations higher than time effect. This showed that the interaction happened instantly when these small molecular were accessed to trypsin.Similarly, we studied the fluorescence spectra changes of trypsin, it is showed the three molecular had caused increasing in intensity and blue shift (except puerarin). The quenching constant Kq is calculated according to the Stern-Volmer equation and the fluorescence effect maybe belong to static quenching.On the other hand, we observed the fluorescence intensity of free camptothecin and found it was higher in the presence of trypsin. This suggests camptothecin do not combine with trypsin in covalent bond, yet formed into a rigidity super molecular compound in other way.The change of trypsin's enzymology quality after the treatment with these alkaloid were also studied,. The results showed that they all inhibit trypsin and the order of inhibition is puerarin> colchicines> camptothecin in which is consistent with the order of fluorescence quenching and enhancement in UV absorption.In brief, this thesis has studied the interactions between three distinctive alkaloids and trypsin by utilizing the means of Spectrum Analysis, thus finding out the following results: the puerarin has the best capacity to make the trypsin lose its Fluorescent, and inhibit its enzyme activation as well colchicine plays the second place and camptothecin does the last one. Based on the above, it might be speculated that these alkaloid and trypsin could form super-molecular compound by inferior linkage between them, causing the Trp of trypsin penetrate form its external surface to internal, and forming position resistance simultaneously. Finally this process triggers some phenomena including the inhibition of Enzyme activation, the loss of fluorescent intensity, blue shift, and so on.