Interaction of α-and β-Oligoarginine-Acids and Amides with Anionic Lipid Vesicles:  A Mechanistic and Thermodynamic Study^†

The interaction of α- and β-oligoarginine amides and acids and of α-polyarginine with anionic lipid vesicles was studied. The β-oligoarginines used were β³-homologues of the α-oligoarginines. Lipid bilayers were composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]) containing 5 mol % pyrene-PG (1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-[phospho-rac-1-glycerol]). Kinetic analysis of the binding process onto large unilamellar POPC/POPG (3:7, molar ratio) vesicles (100 nm diameter) shows biphasic time courses for all tested peptides. The first binding step is fast and takes place within 10 s with no disruption of the membrane as indicated by corresponding calcein release measurements. The second binding phase is slow and occurs within the next 30−300 s with substantial membrane disruption. In this context, β-hexa- and octaarginine amides possess higher second half-times than the β-hexa- and octaarginine acids of the same chain length. Furthermore β-octaarginine amide induces a calcein release approximately twice as large as that of the β-octaarginine acid. Thermodynamic analysis of the binding process, using the complex formation model that assumes that each peptide binds independently to n POPG lipids, reveals apparent binding constants (K_app1) of 5 × 10⁶ − 10⁸ M^-1 and n-values from 3.7 for β-hexaarginine acid up to 24.8 for α-polyarginine. Although the K_app1-values are similar, the number of binding sites clearly depends on the chemical nature of the oligoarginine:  β-oligoarginine amides and α-oligoarginine acids interact with more lipids than β-oligoarginine acids of the same length. Calculation of the electrostatic contribution to the total free energy of binding reveals that for all oligoarginines only 25−30% has electrostatic origin. The remaining 70−75% is nonelectrostatic, corresponding to hydrogen bonding and/or hydrophobic interactions. From the obtained data, a mechanism is suggested by which oligoarginines interact with anionic vesicles:  (1) initial electrostatic interaction that is fast, nonspecific, and relatively weak; (2) nonelectrostatic interaction that is rate-limiting, stronger, and induces bilayer rigidification as well as release of aqueous contents from the vesicles.