Modification of opioid ligand binding in the central and the peripheral nervous system by different buffers

The modification of binding parameters (equilibrium dissociation constant and binding capacity) of three opioid ligands (DADLE, Etorphine and EKC) on bovine adrenal medulla and rat brain membranes have been examined in three buffer systems: Tris-HCl 50 mM, Hepes-NaOH 10 mM and Tes-KOH 10 mM. Major differences of these parameters have been found: Hepes-NaOH provoked a diminution of the apparent number of binding sites, while a concomitant diminution of the KD and Bmax was observed in Tes-KOH buffer. Substitution of counterions in these two buffers produced further changes of binding characteristics: in Hepes buffer we have observed an abolition of 3H DADLE binding, an enhancement of 3H EKC binding and no modification of 3H etorphine binding characteristics. On the contrary an abolition of the specific binding of all three ligands in Tes buffer was found in the bovine adrenal medulla while minor changes were observed in rat brain. It is concluded that, inspite same disadvantages (substitution for bivalent cations and temperature dependence), Tris-HCl is the buffer of choice for the analysis of opioid binding site interactions.