Fluorescence spectroscopic determination of dipyridamole binding on pancreas-1 tumor cell membrane

BACKGROUND: The traditional method to determine the binding sites of inhibitors bound to nucleoside transporters on cell membrane is the radioactive assay. This method suffers from radiolabel instability and the need to dispose of reactive materials. Fluorescence spectroscopy has been increasingly applied to biochemical analysis due to its high sensitivity and selectivity. We describe fluorescence spectroscopy for the determination of the binding sites of dipyridamole bound to nucleoside transporters on pancreas-1 tumor cell membrane. METHODS: Pancreas-1 tumor cell was cultured in Dulbecco's modified Eagle medium under appropriate conditions. The cell plasma membrane was separated from the ultrasonically dissolved cell mixture by gradient centrifugation. After dipyridamole was bound to the cell membrane, the test sample was obtained by ultrasonically dissolving the labeled membrane in 10 mmol/l tris-HCl membrane lysis solution. Under selected experimental conditions, the fluorescence intensity of dipyridamole was determined. RESULTS: At lambda em of 295 nm and lambda em of 485 nm, dipyridamole was proportional to its concentration ranging from 1.0 x 10(-12) to 5.0 x 10(-11) mol/l with a detection limit of 2.8 x 10(-13) mol/l (0.14 pg/ml) at 3sigma. The value of affinity constant of the cell membrane to dipyridamole was 4.7(+/-0.5) x 10(10) l/mol and the average total number of binding sites of a pancreas-1 tumor cell was 1.9(+/-0.2) x 10(6). CONCLUSIONS: The proposed method can be used in the study of binding characters of dipyridamole on the cell membrane.