The molecular characterisation and antimicrobial activity of amidated bovine lactoferrin

Chemical modification of bovine lactoferrin (LF) by amidation increased the net positive charge of the protein and markedly enhanced its activity against most Gram-positive and Gram-negative bacteria tested, but not against Saccharomyces cerevisiae or Penicillium candidum. Bovine lactoferrin was amidated with 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide in the presence of ammonium ions. Mass spectrometric analysis of the modified protein confirmed that amidation converted aspartyl or glutamyl residues to asparaginyl or glutaminyl residues. The changes in net charge and electrophoretic mobility of amidated LF were confirmed by electrophoretic and chromatographic methods. Isoionic point titration was used to determine changes to net charges and ion exchange chromatography results confirmed that the increased positive charge played an important role in the antimicrobial action of the amidated protein. Thus, chemical amidation of LF introduced substantial structural changes in the LF molecule, reflected in amino acid composition and increased net positive charge, and enhanced biological activity.