The Spectrofluorimetric Dihydrofolate Reductase Inhibitor Screening Model For The Establishment And Application

Dihydrofolate reductase (DHFR) is a crucial enzyme in biochemistry. The enzyme has been the subject of extensive study because of its importance in intermediary metabolism and it is the target for a number of chemotherapeutic. The function of this enzyme is to reduce dihydrofolic acid to tetrahydrofolic acid using NADPH as the hydrogen donor (cofactor). Tetrahydrofolic acid is then converted to one-carbon adducts, which are utilized in a number of one-carbon transfers in the bio-synthesis. Recently years, the inhibitor of DHFR and the quantitative structure-activity relationships were widely studied. Therefore , it is necessary to develop a simple method to screen DHFR inhibitors. This paper includes the following contends:In chapter one, the assay methods of activity of enzyme, and the factors of enzyme catalyzed reaction and the application of fluorophotometry in enzyme assay were summarized.In chapter two, a method for fluorophotometry assay of dihydrofolate reductase (DHFR) activity by the fluorescence of dihydrofolate (DHFA) and NADPH was developed. Some reaction conditions have been optimized. The developed model was applied to determination of inhibit rate of some DHFR inhibitors. The method is sensitive and economical and it is suitable for screening DHFR inhibitors.In chapter three, in order to save the experiment cost, and improve the enzyme activity, DHFR has been purified from chicken liver. The concentration of protein, and enzyme activity of each purification procedure were determined. The product can be applied in the next experiment.In chapter four, a method for fluorophotometry assay of chicken liver dihydrofolate reductase (DHFR) activity was developed. The developed method was applied to screening DHFR inhibitors.In chapter five, a method for determination of trace folic acid in milk powder and urine samples by potassium permanganate oxidation-photochemical reaction fluorimetry was studied. In 0.01mol/L hydrochloric acid medium, using potassium permanganate as oxidant and ultraviolet radiation, the fluorescence intensity of folic acid can be greatly increased. The method for pretreatment of samples was also studied. When the sample was purified with diatomite by chromatography, the method can be applied in determining folic acid in milk powder and urine .