The lipid-binding properties of serum apolipoproteins are mediated by class A amphipathic alpha-helices in which basic and acidic amino acid residues tend to be localized at the helix polar-nonpolar boundary and the center of the polar face, respectively. To better understand the role of the interfacial basic residues, the microenvironments and titration characteristics of lysine (Lys) residues have been examined using two 18-residue peptides. The parent 18A molecule (AspTrpLeuLysAlaPheTyrAspLysValAlaGluLysLeuLysGluAlaPhe) binds phospholipid relatively well because of its class A amphipathic helix, whereas 18R, which has the positions of basic and acidic residues reversed, binds to phospholipid relatively weakly. Lys residues were 13C-dimethylated, and NMR spectra were obtained of the peptides complexed with dimyristoylphosphatidylcholine (1/1 (w/w) DMPC/peptide). The four [13C]-epsilon-dimethyl-Lys in 18A gave four resonances at chemical shifts (delta) of 42-43 ppm, whereas only a single resonance at delta 42.70 ppm was observed from the four Lys in 18R. Measurements of delta as a function of pH gave pKa values. The four Lys in 18R had a common pKa of 10.3, indicating that the Lys in the polar face of the amphipathic helix have the pKa expected for noninteracting, fully hydrated groups. In contrast, the four Lys in Ac-18A-NH2 had pKa's in the range 9.4-11.0, indicating that they exist in various microenvironments at the polar-nonpolar boundary of the 18A helix. To examine individual Lys, 18A was synthesized with various Lys selectively labeled. Lys-4, -9, -13, and -15 in Ac-18A-NH2 associated with DMPC had pKa values of 11.0, 9.4, 9.4, and 10.3, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)