Characterization of an angiotensin II-fluorescamine derivative.
The coupling of fluorescamine (4-phenylspiro[furan-2(3H), 1'-PHTHALAN]-3,3'dione) to angiotensin II to form a fluorescent derivative was studied. Complete reaction of the peptide below concentrations of 10- minus 4 M could be achieved with a fluorescamine concentration of 0-3 mg ml- minus 1 of acetone at pH 8-3, and the lowest concentration detectable by fluorescence spectroscopy was 100 pmol ml- minus 1. The derivative, as prepared did not react with ninhydrin, and no fluorescence was generated when fluorescamine was reacted with (1-Sar)-ATII. These data suggest that fluorescence is generated only through the coupling of fluorescamine to the N-terminal primary amine of ATII. The ATII-fluorescamine derivative has the same intrinsic activity on the contraction of rat colon (elevenfold loss of affinity), and on the release of fluorogenic corticosteroids from bovine adrenal cortical slices (sixfold loss of affinity) compared to ATII. Water-hydrolysed fluorescamine and Asp-fluorescamine did not contract rat colon preparations; the contractile response to ATII-fluorescamine was blocked by (8-Leu)-ATII, a specific ATII antagonist. These findings suggest that theATII fluorophore shares a common receptor site with the native octapeptide. The rate loss of biological activity of the ATII-fluorescamine derivative was appreciably lower than that observed for ATII. The present study suggests that the ATII-fluorescamine derivative can be substituted for radioactively-labelled ATII for use in a variety of applications.