Models of protein translocation and secretion will not be complete without details of the mechanism of lipid bilayer insertion. The study of spontaneous hydrophobic peptide interactions with model membrane systems has been hindered by their very low solubility in aqueous solutions. A novel protocol has been developed that enables the site-specific (N-terminus) attachment of hydrophobic peptides to a water-soluble carrier protein [bovine pancreatic trypsin inhibitor (BPTI)] using a heterobifunctional crosslinker (SPDP). In this initial study H-(Ala)20-Tyr-Cys-CONH2 and H-(Ala)10-Tyr-Cys-CONH2 were selected as hydrophobic peptides, since alanine is the simplest alpha-helix-forming amino acid, and the peptides as alpha-helices are just long enough to span the lipid bilayer and monolayer, respectively. The carrier protein was treated with sigma-methylisourea, which resulted in the guanidination of the four lysine epsilon-amine groups. The chemical modification of BPTI to give G-BPTI allowed the attachment of SPDP specifically to the free N-terminal alpha-amine group. The peptides were synthesized with a C-terminal cysteine moiety, allowing the site-specific cross-linking of the peptides to the N-terminus. In order to prevent peptide aggregation, the synthetic peptides were cleaved from the preparative resin in detergent and cross-linked to G-BPTI. After cross-linking, the detergent was removed from the mixture by gel filtration employing propionic and formic acids in the mobile phase. The detergent-free, peptide--G-BPTI conjugates were subsequently purified by reversed-phase HPLC. The interaction parameters of the two semisynthetic proteins with large unilamellar vesicles were determined by ultracentrifugation of the equilibrated vesicle--protein mixtures. For comparison, the same semisynthetic proteins were reconstituted into lipid vesicles using an octyl glucoside dilution technique. The incorporation and reconstitution data proved to be quite similar. The results indicated that (Ala)20--G-BPTI interacted with LUV to form a stable complex and behaved as a membrane protein in reconstituted bilayer systems. (Ala)10--G-BPTI, however, remained in the aqueous phase in both bilayer systems. The thermodynamic interaction data are compared to the theoretical values of total free energy changes calculated for the incorporation of model hydrophobic alpha-helices. In addition, the solubility and stability of the hydrophobic peptides, both in the aqueous phase and membrane-bound, were studied by cleaving the disulfide bond linking the peptides to G-BPTI using dithiothreitol. Molecular sieve chromatography was used to evaluate the state of self-association of the semisynthetic proteins in aqueous solutions.