Preparation of a Photoactivable Ligand for Bradykinin Receptors

ABSTRACT Most of physiological effects of bradykinin is due to its effect on bradykinin B2 receptors. There is no reports of preparation of a photoactivatable analogue of bradykinin reactive with sloubilized bradykinin B2 recpetors. Photoactivatable radioactive ligands are powerful tools in purification of receptors. In this study we report preparation of a photoactivatable iodinatable analogue of bradykinin reactive with soluble bradykinin B2 receptors. For conjugation with lysyl-bradykinin (LBK) a photoactivatable compound sulfosuccimidyl 2-(pazidosalicylamido ethyl-1,3-dithiopropionate (SASD) was used. Eigtheen hours after incubation in safe light(25 watt red light) LBK-SASD cojugate was prepared. HPLC experiments showed that retention time for conjugate on the HPLC column was 26 minutes. This was different from retention time of starting materials (for LBK 17 and for SASD 24 minutes). Also inhibition studies showed that LBK-SASD conjugate is capable of inhibiting binding of an antibody against bradykinin to ELISA plates coated with LBK-glut-BSA.This shows that in comparison with SASD and LBK the conjugate retained its immunoreactivity. Soluble binding assay showed that the LBK-SASD conjugate is reactive with solubilized bovine uterin myometrium bradykinin B2 receptors. With the addition of I125 to the conjugate it is possible to radiolabel the receptors. In the first step radioactive conjugate is reacted with the solubilized receptor, then upon exposure of the light a covalent bond is formed between receptor and the ligand. With monitoring the radioactivity it will be possible to check the success of the purification procedures.