Ortho-aminobenzoic acid-labeled bradykinins in interaction with lipid vesicles: fluorescence study.
The peptide hormone bradykinin (BK) (Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)) and its shorter homolog BK(1-5) (Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)) were labeled with the extrinsic fluorescent probe ortho-aminobenzoic acid (Abz) bound to the N-terminal and amidated in the C-terminal carboxyl group (Abz-BK-NH(2) and Abz-BK(1-5)-NH(2)). The fragment des-Arg(9)-BK was synthesized with the Abz fluorescent probe attached to the 3-amino group of 2,3-amino propionic acid (DAP), which positioned the Abz group at the C-terminal side of BK sequence, constituting the peptide des-Arg(9)-BK-DAP(Abz)-NH(2). The spectral characteristics of the probe were similar in the three peptides, and their fluorescent properties were monitored to study the interaction of the peptides with anionic vesicles of dimyristoylphosphatidylglycerol (DMPG). Time-resolved fluorescence experiments showed that the fluorescence decay of the peptides was best described by double-exponential kinetics, with mean lifetimes values around 8.0 ns in buffer pH 7.4 that increased about 10% in the presence of DMPG vesicles. About a 10-fold increase, compared with the values in aqueous solution, was observed in the steady-state anisotropy in the presence of vesicles. A similar increase was also observed for the rotational correlation times obtained from time-resolved anisotropy decay profiles, and related to the overall tumbling of the peptides. Equilibrium binding constants for the peptide-lipid interaction were examined monitoring anisotropy values in titration experiments and the electrostatic effects were evaluated through Gouy-Chapman potential calculations. Without corrections for electrostatic effects, the labeled fragment Abz-BK(1-5)-NH(2) presented the major affinity for DMPG vesicles. Corrections for the changes in peptide concentration due to electrostatic interactions suggested higher affinity of the BK fragments to the hydrophobic phase of the bilayer.
Turchiello
R F
RF
Instituto de Fisica, Universidade de São Paulo, Brasil.
Lamy-Freund
M T
MT
Hirata
I Y
IY
Juliano
L
L
Ito
A S
AS
D001920:Bradykinin / Q000031:analogs & derivatives* / Q000737:chemistry; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry; D066298:In Vitro Techniques; D008081:Liposomes; D010446:Peptide Fragments / Q000737:chemistry; D010715:Phosphatidylglycerols; D055672:Static Electricity; D062367:ortho-Aminobenzoates / Q000737:chemistry
D005456:Fluorescent Dyes; D008081:Liposomes; D010446:Peptide Fragments; D010715:Phosphatidylglycerols; D062367:ortho-Aminobenzoates; C002773:dimyristoylphosphatidylglycerol; D001920:Bradykinin