Production, characterization, and elucidation of structure-function relationship of lactoferricin and other peptides derived from food-grade bovine lactoferrin

Lactoferricin (Lfcin), a cationic antimicrobial peptide, was purified by peptic digestion of food grade bovine lactoferrin (LF) followed by fractionation on an industrial grade cation exchange resin with stepwise salt gradient elution. A 2-step process using competitive displacement cation exchange chromatography led to 35% recovery of peptides with masses of 3124 and 3196 Da corresponding to Lfcin. Other cationic peptides produced concurrently with Lfcin were tentatively identified. Varying iron saturation levels of LF had no effect on the production of Lfcin. Two approaches were used to understand how Lfcin acts as an antimicrobial agent. Homology Similarity Analysis (HSA) was used to evaluate the impact of peptide pattern similarities of various Lfcin derivatives on their effects as antimicrobial agents. Helical property of residues 4-9 in the Lfcin sequence was the most important in determining the antimicrobial activity of Lfcin against Escherichia coli, followed by cationic charge pattern of residues 4-9 and residues 1-3. Raman spectroscopy in the C-H stretching (2800-3000 cm⁻¹) region was used to study interactions between Lfcin and bacterial membrane models. Presence of Lfcin in 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) multilamellar liposomes restricted the lateral chain-chain interaction along the acyl chain throughout the temperature range examined, and increased the main transition temperature (Tm) from 38-40°C. Lfcin had little effect on 1,2-dipalmitoyl-sn-glycerol-3-[phosphor-rac-(1-glycerol)] (DPPG) liposomes at temperatures below the Tm. In contrast, mobility of the acyl chains in 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine (DPPE) was increased in the presence of Lfcin at temperatures below 32°C. Although Lfcin had little effect on the zwitterionic unsaturated phospholipids, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolamine (POPE), Lfcin lowered the lateral chain-chain interaction along the acyl chains of negatively charged 1-palmitoyl-2-oleoyl-sn-glycerol-3-[phosphor-rac-(1-glycerol)] (POPG). Raman spectral analyses also showed that the removal of arginine promoted interactions of Lfcin derivatives with hydrophobic acyl chains and methyl ends of negatively charged DPPG liposomes. Furthermore, addition of two extra tryptophan residues in Lfcin derivatives stabilized acyl chains and methyl ends in DPPE liposomes. HSA and Raman spectroscopy are two useful tools for understanding the antimicrobial mechanisms of Lfcin and will facilitate the application and utilization of Lfcin as a naturally occurring antimicrobial agent.Land and Food Systems, Faculty ofGraduat