Ortho-aminobenzoic acid as a fluorescent probe for the interaction between peptides and micelles

Ortho-aminobenzoic acid (o-Abz) has been used as a fluorescent probe in internally quenched fluorescent peptides for continuous protease assays. We investigated the fluorescent properties of the probe in order to verify if it can be used to monitor the interaction of peptides with micelles. Abz-aminoacyl-monomethyl amides (Abz-Xaa-NHCH(3), where Xaa=Arg, Phe, Leu and Glu) were synthesized. Quantum yield, spectral position, anisotropy and lifetime decay were analyzed in the presence and absence of sodium dodecyl sulfate (SDS) micelles. Significant changes in the fluorescence parameters were observed for Abz-Arg-NHCH(3) in comparison to Abz-Glu-NHCH(3), indicating a strong electrostatic component in the compound's interaction with the negative charged micelles. The change in fluorescence parameters, observed when the probe is bound to hydrophobic amino acids Abz-Phe-NHCH(3) and Abz-Leu-NHCH(3), is probably due to insertion of those compounds into micelles. Abz-NHCH(3) fluorescence is less affected by the presence of micelles, indicating that the occurrence of interaction is dependent on the properties of the amino acid to which the fluorophore is attached. The quenching data with acrylamide confirmed these results. Titration curves allowed the estimation of association constants between Abz compounds and SDS, according to a single partition model. Although the results cannot be strictly applied to the titration with charged compounds, it was verified that the association constant for the isolated Abz-NHCH(3) is significantly lower than those for Abz-Phe-NHCH(3) and Abz-Leu-NHCH(3). It is concluded that the Abz group is a sensitive and convenient fluorescent probe to monitor peptide binding to amphiphilic aggregates. That conclusion is supported by measurements with the peptide Abz-Leu-Arg-Phe-NH(2).