ON THE INTERACTION OF DIPYRIDAMOLE DERIVATIVES WITH BOVINE SERUM-ALBUMIN - A FLUORESCENCE STUDY

Interaction of several derivatives of the coronary vasodilator dipyridamole (DIP) with the transport protein bovine serum albumin (BSA) has been studied using fluorescence spectroscopy. Analysis of emission spectra and fluorescence intensity of DIP derivatives in solution allowed the determination of binding parameters and stoichiometry. The derivatives studied were RA14, RA39, RA25 and RA47. They have the same pyrimido-pyrimidine structure and different substituents, so our aim is to obtain information on chemical structure-biological activity relationship. Measurements were performed at pH 7.0 and pH 5.0 in order to analyze the effect of protonation of the DIP molecule on binding. The association constants, K-a, obtained from emission intensity titration, were in the range (0.2-10.0) x 10(3) M(-1) and the number of binding sites was close to one. Static anisotropy of fluorescence was also used to monitor the binding. In aqueous solution the anisotropy is essentially zero; binding to the protein leads to an increase in the values of r(0) to 0.2-0.3. Data were fitted to a simple binding model and maximal values of r and K-a were obtained showing a good agreement with the parameters obtained from emission intensity measurements. DIP, RA14 and RA39 are tightly bound to the protein displaying a larger anisotropy than RA47 and RA25, suggesting that these last two derivatives form a weak binding in the surface of the protein. The order for the values of association constants is RA25 < RA47 < DIP = RA14, which is in accordance with the reported biological activity. Fluorescence quenching using acrylamide and iodide allowed further localization of the drug in the protein molecule. At pH 7.0 the derivatives are more protected in the interior of the protein, while at pH 5.0 they become more exposed and susceptible to a more efficient quenching, especially by iodide.