Effect of gamma radiation on the structural and biological properties of angiotensin II.

PURPOSE: The vasoactive octapeptide hormone angiotensin II (DRVYIHPF, AngII) was selected as the target of this2investigation, which was aimed at determining the effect of gamma radiation on peptide structure and biological activity. MATERIALS AND METHODS: Radiation doses ranging from 1-15 kGy were applied to samples of purified AngII. RESULTS: The measured amount of remaining native hormone decreased non-linearly as the gamma radiation dose increases. Amino acid analysis of these irradiated peptide solutions demonstrated similar, simultaneous modifications of Phe8 and His6 residues along with the increase in the radiation dose. This structural variation of the vasoactive peptide closely resembled the decreasing process of the biological potencies of irradiated peptide solutions in rat uterus and guinea pig ileum muscle preparations. CONCLUSIONS: These findings suggest that investigating the effect of gamma radiation on small model molecules such as peptides could be of value for further extending this type of study to other physiologically relevant macromolecules such as proteins. Of note, this unique approach could also be useful in generating different types of peptide analogs (after purification) for application in future classical structure-function studies.

Functional assessment of angiotensin II and bradykinin analogues containing the paramagnetic amino acid TOAC.

This study characterized pharmacologically the functional responses to agonists angiotensin II (AngII) and bradykinin (BK) derivatives containing the TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) spin label at the N-terminal (TOAC1-AngII and TOAC0-BK) and internal (TOAC3-AngII and TOAC3-BK) positions of these vasoactive peptides. Affinity constants of the ligands for AT1 and B2 receptors were evaluated in vitro by binding assays and biological effects by extracellular acidification rates and in vivo by blood pressure responses. In contrast to internally labeled analogues (TOAC3-AngII or TOAC3-BK), the TOAC1-AngII and TOAC0-BK derivatives dose-dependently increased the extracellular acidification rate in adherent cultured Chinese hamster ovary (CHO) cells expressing AT1 or B2 receptors, respectively. In addition, TOAC(1)-AngII induced an increase in blood pressure when injected intravenously in awaken rats although with a potency four times smaller when compared to native AngII. Similarly to BK, TOAC0-BK dose-dependently decreased blood pressure when injected intra-arterially in rats with a lower potency when compared to the native peptide. On the contrary, TOAC3-AngII or TOAC3-BK did not provoke any alteration in blood pressure levels. In summary, our results confirmed that the insertion of TOAC-probe in the N-terminal region of peptides does not significantly modify the affinity or biological activity in vitro and in vivo conditions and could be an important tool to evaluate peptide-receptor interaction mechanism. Conversely, possibly due to the unique bend-inducing property of the cyclic TOAC probe, its insertion at position 3 in both AngII and BK structures seems to restrict the interaction and the activation of the AT1 and B2 receptors.