Bulky aromatic amino acids increase the antibacterial activity of 15-residue bovine lactoferricin derivatives.

A model peptide, FKCRRWQWRMKKLGA, residues 17-31 of bovine lactoferricin, has been subjected to structure-antibacterial activity relationship studies. The two Trp residues are very important for antibacterial activity, and analogue studies have demonstrated the significance of the size, shape and aromatic character of the side chains. In the current study we have replaced Trp residues in the model peptide with bulky aromatic amino acids to elucidate further the importance of size and shape. The counterproductive Cys residue in position 3 was also replaced by these aromatic amino acids. The largest aromatic amino acids employed resulted in the most active peptides. The peptides containing these hydrophobic residues were generally more active against Staphylococcus aureus than against Escherichia coli, indicating that the bacterial specificity as well as the antibacterial efficiency can be altered by employing large hydrophobic aromatic amino acid residues.     

Enhanced antitumour activity of 15-residue bovine lactoferricin derivatives containing bulky aromatic amino acids and lipophilic N-terminal modifications.

In a structure-antibacterial activity relationship study of a peptide fragment of bovine lactoferricin consisting of FKCRRWQWRMKKLGA (LFB 17-31), it was revealed that the two Trp residues were important for antibacterial activity. It has further been demonstrated that the size, shape and the aromatic character of the side chains were even more important than the Trp itself. In this study the antitumour effect of a series of LFB 17-31 derivatives are reported, in which the two Trp residues in position 6 and 8 were replaced with the larger non-coded aromatic amino acids Tbt, Tpc, Bip and Dip. The counterproductive Cys in position 3 was also substituted with these larger aromatic residues. In addition, the effect of introducing lipophilic groups of different size and shape in the N-terminal of the LFB 17-31 sequence was addressed. The resulting peptide derivatives were tested for activity against three human tumour cell lines and against normal human umbilical vein endothelial cells and fibroblasts. High antitumour activity by several of the peptides demonstrated that Trp successfully could be substituted by the bulky aromatic residues, and peptides containing the large and rigid Tbt residue in position 6 and/or 8 in LFB 17-31 were the most active candidates. The antitumour effect was even more increased by the Tbt-modified peptides when the three counterproductive amino acids Cys3, Gln7 and Gly14 were replaced by Ala. Enhanced antitumour activity was also obtained by modifying the N-terminal of LFB 17-31 with either long-chained fatty acids or bulky moieties. Thus, our results revealed that the size and shape of the lipophilic groups and their position in the peptide sequence were important for antitumour activity.     

The antibiotic activity of cationic linear amphipathic peptides: lessons from the action of leucine/lysine copolymers on bacteria of the class Mollicutes.

Peptides composed of leucyl and lysyl residues ('LK peptides') with different compositions and sequences were compared for their antibacterial activities using cell wall-less bacteria of the class Mollicutes (acholeplasmas, mycoplasmas and spiroplasmas) as targets. The antibacterial activity of the amphipathic alpha-helical peptides varied with their size, 15 residues being the optimal length, independent of the membrane hydrophobic core thickness and the amount of cholesterol. The 15-residue ideally amphipathic alpha helix with a +5 positive net charge (KLLKLLLKLLLKLLK) had the strongest antibacterial activity, similar to that of melittin. In contrast, scrambled peptides devoid of amphipathy and the less hydrophobic beta-sheeted peptides [(LK)nK], even those 15-residue long, were far less potent than the helical ones. Furthermore, the growth inhibitory activity of the peptides was correlated with their ability to abolish membrane potential. These data are fully consistent with a predominantly flat orientation of LK peptides at the lipid/water interface and strongly supports that these peptides and probably the linear polycationic amphipathic defence peptides act on bacterial membranes in four main steps according to the 'carpet' model: (a) interfacial partitioning with accumulation of monomers on the target membrane (limiting step); (b) peptide structural changes (conformation, aggregation, and orientation) induced by interactions with the lipid bilayer (as already shown with liposomes and erythrocytes); (c) plasma membrane permeabilization/depolarization via a detergent-like effect; and (d) rapid bacterial cell death if the extent of depolarization is maintained above a critical threshold.     

Effects of tryptophan residues of porcine myeloid antibacterial peptide PMAP-23 on antibiotic activity.

PMAP-23 is a 23-residue antimicrobial peptide from porcine myeloid cells. In order to determine the effects of two Trp residues in positions 7 and 21 of PMAP-23 on antibacterial activity and phospholipid vesicle interacting property, two analogues in which Ala is substituted for Trp residue in position 7 or 21 were synthesized. A(21)-PMAP-23 exhibited reduced antibacterial activity and phospholipid vesicle disrupting activity when compared to those of PMAP-23 and A(7)-PMAP-23. PMAP-23 readily interacted with model lipid membrane and induced membrane destabilization. Therefore antibacterial activity induced by PMAP-23 is due to the interaction of cell membrane with peptide followed by membrane perturbation. A significant structural change on the SDS micelle was not found by Ala substitution of the Trp residue of PMAP-23. Also, there is a good correlation between hydrophobic interaction on RP-HPLC, expressed as retention time on RP-HPLC, and antibacterial activity. The vesicle titration experiment indicated that Trp residues located at near C-terminus are accessible to hydrophobic tail of phospholipid vesicle. This result suggests that the C-terminal end of PMAP-23 penetrates into the lipid bilayer in the course of the interaction with phospholipid membranes and is important for its antibacterial activity.     

Important structural features of 15-residue lactoferricin derivatives and methods for improvement of antimicrobial activity.

This review focuses on important structural features affecting the antimicrobial activity of 15-residue derivatives of lactoferricins. Our investigations are based on an alanine-scan of a 15-residue bovine lactoferricin derivative that revealed the absolute necessity of two tryptophan residues for antimicrobial activity. This "tryptophan-effect" was further explored in homologous derivatives of human, caprine, and porcine lactoferricins by the incorporation of one additional tryptophan residue, and by increasing the content of tryptophan in the bovine derivative to five residues. Most of the resulting peptides display a substantial increase in antimicrobial activity. To identify which molecular properties make tryptophan so effective, a series of bovine lactoferricin derivatives were prepared containing non-encoded unnatural aromatic amino acids, which represented various aspects of the physicochemical nature of tryptophan. The results clearly demonstrate that tryptophan is not unique since most of the modified peptides were of higher antimicrobial potency than the native peptide. The size and three-dimensional shape of the inserted "super-tryptophans" are the most important determinants for the high antimicrobial activity of the modified peptides. This review also describes the use of a "soft-modeling" approach in order to identify important structural parameters affecting the antimicrobial activity of modified 15-residue murine lactoferricin derivatives. This QSAR-study revealed that the net charge, charge asymmetry, and micelle affinity of the peptides were the most important structural parameters affecting their antimicrobial activity.     

Structural studies and model membrane interactions of two peptides derived from bovine lactoferricin.

The powerful antimicrobial properties of bovine lactoferricin (LfcinB) make it attractive for the development of new antimicrobial agents. An 11-residue linear peptide portion of LfcinB has been reported to have similar antimicrobial activity to lactoferricin itself, but with lower hemolytic activity. The membrane-binding and membrane-perturbing properties of this peptide were studied together with an amidated synthetic version with an added disulfide bond, which was designed to confer increased stability and possibly activity. The antimicrobial and cytotoxic properties of the peptides were measured against Staphylococcus aureus and Escherichia coli and by hemolysis assays. The peptides were also tested in an anti-cancer assay against neuroblastoma cell lines. Vesicle disruption caused by these LfcinB derivatives was studied using the fluorescent reporter molecule calcein. The extent of burial of the two Trp residues in membrane mimetic environments were quantitated by fluorescence. Finally, the solution NMR structures of the peptides bound to SDS micelles were determined to provide insight into their membrane bound state. The cyclic peptide was found to have greater antimicrobial potency than its linear counterpart. Consistent with this property, the two Trp residues of the modified peptide were suggested to be embedded deeper into the membrane. Although both peptides adopt an amphipathic structure without any regular alpha-helical or beta-sheet conformation, the 3D-structures revealed a clearer partitioning of the cationic and hydrophobic faces for the cyclic peptide.     

Tryptophan- and arginine-rich antimicrobial peptides: Structures and mechanisms of action

Antimicrobial peptides encompass a number of different classes, including those that are rich in a particular amino acid. An important subset are peptides rich in Arg and Trp residues, such as indolicidin and tritrpticin, that have broad and potent antimicrobial activity. The importance of these two amino acids for antimicrobial activity was highlighted through the screening of a complete combinatorial library of hexapeptides. These residues possess some crucial chemical properties that make them suitable components of antimicrobial peptides. Trp has a distinct preference for the interfacial region of lipid bilayers, while Arg residues endow the peptides with cationic charges and hydrogen bonding properties necessary for interaction with the abundant anionic components of bacterial membranes. In combination, these two residues are capable of participating in cation-π interactions, thereby facilitating enhanced peptide-membrane interactions. Trp sidechains are also implicated in peptide and protein folding in aqueous solution, where they contribute by maintaining native and nonnative hydrophobic contacts. This has been observed for the antimicrobial peptide from human lactoferrin, possibly restraining the peptide structure in a suitable conformation to interact with the bacterial membrane. These unique properties make the Arg- and Trp-rich antimicrobial peptides highly active even at very short peptide lengths. Moreover, they lead to structures for membrane-mimetic bound peptides that go far beyond regular α-helices and β-sheet structures. In this review, the structures of a number of different Trp- and Arg-rich antimicrobial peptides are examined and some of the major mechanistic studies are presented.     

Tryptophan- and arginine-rich antimicrobial peptides: structures and mechanisms of action

Antimicrobial peptides encompass a number of different classes, including those that are rich in a particular amino acid. An important subset are peptides rich in Arg and Trp residues, such as indolicidin and tritrpticin, that have broad and potent antimicrobial activity. The importance of these two amino acids for antimicrobial activity was highlighted through the screening of a complete combinatorial library of hexapeptides. These residues possess some crucial chemical properties that make them suitable components of antimicrobial peptides. Trp has a distinct preference for the interfacial region of lipid bilayers, while Arg residues endow the peptides with cationic charges and hydrogen bonding properties necessary for interaction with the abundant anionic components of bacterial membranes. In combination, these two residues are capable of participating in cation-pi interactions, thereby facilitating enhanced peptide-membrane interactions. Trp sidechains are also implicated in peptide and protein folding in aqueous solution, where they contribute by maintaining native and nonnative hydrophobic contacts. This has been observed for the antimicrobial peptide from human lactoferrin, possibly restraining the peptide structure in a suitable conformation to interact with the bacterial membrane. These unique properties make the Arg- and Trp-rich antimicrobial peptides highly active even at very short peptide lengths. Moreover, they lead to structures for membrane-mimetic bound peptides that go far beyond regular alpha-helices and beta-sheet structures. In this review, the structures of a number of different Trp- and Arg-rich antimicrobial peptides are examined and some of the major mechanistic studies are presented.     

Comparison of NMR structures and model-membrane interactions of 15-residue antimicrobial peptides derived from bovine lactoferricin.

LFB (FKCRRWQWRMKKLGA-HN2) is a 15-residue linear antimicrobial peptide derived from bovine lactoferricin, which has antimicrobial activity similar to that of the intact 25-residue disulfide-cyclized peptide. Previous alanine-scan studies, in which all of the residues in LFB were individually replaced with Ala, showed that the 2 tryptophan (Trp) residues of LFB were crucial to its antimicrobial activity. When either Trp6 or Trp8 was replaced with Ala (LFBA6 and LFBA8, respectively), these 2 peptides were almost devoid of antimicrobial activity. We determined the structures of LFB, LFBA6, and LFBA8 bound to membrane-mimetic SDS micelles using NMR spectroscopy, and studied their interactions with different phospholipid-model membranes. The membrane interactions of LFB exhibited little correlation with its antimicrobial activity, suggesting that the mechanism of action of LFB involves intracellular targets. However, the much higher antimicrobial activity of LFB compared with LFBA6 and LFBA8 might result, in part, from the formation of energetically favorable cation-pi interactions observed only in LFB. Information about the importance of Arg and Trp cation-pi interactions will provide insight for the future design of potent antimicrobial peptidomimetics.     

Determination of the disulfide array in sapecin, an antibacterial peptide of Sarcophaga peregrina (flesh fly)

Sapecin is a 40-residue peptide containing 6 half-cystine residues. The disulfide structure of sapecin was determined by sequencing cystine-containing peptides obtained by digesting sapecin with thermolysin. Results showed that sapecin has a vortical structure fixed by 3 disulfide bonds between cysteine residues 3 and 30, 16 and 36, and 20 and 38, respectively, and that these disulfide bonds are essential for its antibacterial activity.     

Biological activities of C-terminal 15-residue synthetic fragment of melittin: design of an analog with improved antibacterial activity

Melittin, the 26-residue predominant toxic peptide from bee venom, exhibits potent antibacterial activity in addition to its hemolytic activity. The synthetic peptide of 15 residues corresponding to its C-terminal end (MCF), which encompasses its most amphiphilic segment, is now being shown to possess antibacterial activity about 5-7 times less compared to that of melittin. MCF, however, is 300 times less hemolytic. An analog of MCF, MCFA, in which two cationic residues have been transpositioned to the N-terminal region from the C-terminal region, exhibits antibacterial activity comparable to that of melittin, but is only marginally more hemolytic than MCF. The biophysical properties of the peptides, like folding and aggregation, correlate well with their biological properties.     

Hydrophobic Effects on Antibacterial and Channel-forming Properties of Cecropin A–Melittin Hybrids

The design of cecropin–melittin hybrid analogues is of interest due to the similarities in the structure of the antimicrobial peptides cecropin and melittin but differences in their lytic properties. We suspected that a hydrophobic residue in position 2 of milittin (Ile⁸ in the hybrid) plays an important role in the activity of the 15-residue hybrid, KWKLFKKIGAVLKVL-NH₂, [CA(1–7)M(2–9)NH₂] and have now examined its role in the analogue toward five test bacteria. Deletion of Ile⁸ reduced activity, and it was not restored by lengthening to 15 residues by addition of another threonine at the C-terminus. Replacement of Ile⁸ by a hydrophobic leucine maintained good activity and Ala⁸ was equally active for four organisms, although less active against Staphylococcus aureus. Replacement by the hydrophilic Ser⁸ strongly reduced potency against all five organisms. Deletion of Leu¹⁵ decreased activity, but addition of Thr¹⁶ maintained good activity. The presence of hydrophobic residues appears to have a significant effect on the process of antibacterial activity. These peptide analogues showed voltage-dependent conductance changes and are capable of forming ion-pores in planar lipid bilayers. The antibacterial action of the peptides is thought to be first an ionic interaction with the anionic phosphate groups of the membrane followed by interaction with the hydrocarbon core of the membrane and subsequent reorientation into amphipathic α-helical peptides that form pores (ion-channels), which span the membrane. The analogue also showed an increase in α-helicity with an increase in hexafluoro 2-propanol concentration.     

Mutational analysis of the role of tryptophan residues in an antimicrobial peptide

Antimicrobial peptides belonging to the pediocin-like family of bacteriocins (class IIa bacteriocins) produced by lactic acid bacteria contain several tryptophan residues that are highly conserved. Since tryptophan residues in membrane proteins are often positioned in the membrane-water interface, we hypothesized that Trp residues in bacteriocins could be important determinants of the structure of membrane-bound peptides and of anti-microbial activity. To test this hypothesis, the effects of mutating each of the 3 tryptophan residues (Trp18, Trp33, and Trp41) in the 43-residue pediocin-like bacteriocin sakacin P were studied. Trp18 and Trp33 are located at each end of an amphihilic alpha-helix, whereas Trp41 is near the end of an unstructured C-terminal tail. Replacement of Trp33 with the hydrophobic residues Leu and Phe had marginal effects on activity, whereas replacement with the more polar Tyr and Arg reduced activity 10-20 and 500-1000 times, respectively, indicating that Trp33 and the C-terminal part of the helix interact with the hydrophobic core of the membrane. Any mutation of Trp18 and Trp41 reduced activity, indicating that these two residues play unique roles. Substitutions with other aromatic residues were the least deleterious, indicating that both Trp18 and Trp41 interact with the membrane-water interface. The suggested locations of the three Trp residues are compatible with a structural model in which the helix and the C-terminal tail form a hairpin-like structure, bringing Trp18 and Trp41 close to each other in the interface, and placing Trp33 in the hydrophobic core of the membrane. Indeed, the deleterious effect of the W18L and W41L mutations could be overcome by stabilizing the hairpin-like structure by introduction of a disulfide bridge between residues 24 and 44. These results provide a basis for a refined structural model of pediocin-like bacteriocins and highlight the unique role that tryptophan residues can play in membrane-interacting peptides.     

The effects of charge and lipophilicity on the antibacterial activity of undecapeptides derived from bovine lactoferricin.

We have investigated the effects of charge and lipophilicity on the antibacterial activity of an undecapeptide (FKCRRWQWRMK) derived from the sequence of bovine lactoferricin. We prepared ten analogues that were modified by the incorporation of Ala, Tyr, Trp, Met and Arg residues, which are amino acids known to be important for the antibacterial activity of longer derivatives of lactoferricins. All undecapeptides contained the native Trp residues in positions 6 and 8, and the Arg residues in positions 5 and 9. Generally, the Gram-positive bacterium Staphylococcus aureus was more susceptible to these undecapeptides than the Gram-negative bacteria, and a higher antibacterial activity was observed against Escherichia coli than against Pseudomonas aeruginosa. The only exception was the peptide Undeca 9 (RRWYRWAWRMR-NH2), which was almost equally active against all three test strains, displaying minimal inhibitory concentrations of 10 microg/ml (5.8 microM), 7.5 microg/ml (4.4 microM) and 5 microg/ml (2.9 microM) against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, respectively. The peptides Undeca 6 (YRAWRWAWRWR-NH2) and Undeca 7 (YRMWRWAWRWR-NH2) were the two most active undecapeptides against Staphylococcus aureus, both displaying a minimal inhibitory concentration of 2.5 microg/ml (1.5 microM). The study showed that a level was reached in which undecapeptides having a net charge above +4 and containing three or four Trp residues all displayed a high antibacterial activity. All undecapeptides prepared were essentially non-haemolytic, but undecapeptides containing more than three Trp residues displayed 50% haemolysis of human red blood cells at concentrations above 400 microg/ml (>230 microM).     

Human defensin 5 disulfide array mutants: disulfide bond deletion attenuates antibacterial activity against Staphylococcus aureus

Human α-defensin 5 (HD5, HD5(ox) to specify the oxidized and disulfide linked form) is a 32-residue cysteine-rich host-defense peptide, expressed and released by small intestinal Paneth cells, that exhibits antibacterial activity against a number of Gram-negative and -positive bacterial strains. To ascertain the contributions of its disulfide array to structure, antimicrobial activity, and proteolytic stability, a series of HD5 double mutant peptides where pairs of cysteine residues corresponding to native disulfide linkages (Cys(3)-Cys(31), Cys(5)-Cys(20), Cys(10)-Cys(30)) were mutated to Ser or Ala residues, overexpressed in E. coli, purified, and characterized. A hexa mutant peptide, HD5[Ser(hexa)], where all six native Cys residues are replaced by Ser residues, was also evaluated. Removal of a single native S-S linkage influences oxidative folding and regioisomerization, antibacterial activity, Gram-negative bacterial membrane permeabilization, and proteolytic stability. Whereas the majority of the HD5 mutant peptides show low micromolar activity against Gram-negative E. coli ATCC 25922 in colony counting assays, the wild-type disulfide array is essential for low micromolar activity against Gram-positive S. aureus ATCC 25923. Removal of a single disulfide bond attenuates the activity observed for HD5(ox) against this Gram-positive bacterial strain. This observation supports the notion that the HD5(ox) mechanism of antibacterial action differs for Gram-negative and Gram-positive species [Wei et al. (2009) J. Biol. Chem. 284, 29180-29192] and that the native disulfide array is a requirement for its activity against S. aureus.     

Addition and omission analogs of the 13-residue antibacterial and hemolytic peptide PKLLKTFLSKWIG: structural preferences, model membrane binding and biological activities.

The consequences of selective addition or deletion of polar amino acids in a 13-residue antibacterial peptide PKLLKTFLSKWIG on structure, membrane binding and biological activities have been investigated. The variants generated are (a) S and T residues replaced by K, (b) S and T residues deleted individually and together, (c) introduction of two additional K and (d) deletion of L and L with T. In the aqueous environment all the peptides were unordered. In trifluoroethanol, the spectra of peptides belonging to groups (a-c) suggest distorted helical conformation. Peptides in group (d) appear to adopt beta-sheet conformation. The peptides bind to zwitterionic and negatively charged lipid vesicles, although to different extents. With the exception of peptides in group (d), all the other peptides exhibited comparable antibacterial activity against Escherichia coli and Staphylococcus aureus. However, the changes made in the peptides in groups (a-c) resulted in reduction of hemolytic activity compared to the parent peptide. Extent of binding to lipid vesicles composed of phosphatidylcholine and cholesterol appears to correlate with hemolytic activity. It appears that polar and charged residues play a major role in modulating the biological activities of the 13-residue peptide PKLLKTFLSKWIG. The 11-residue peptide-like PKLLKFLKWIG has selective antibacterial activity. Thus, by judicious engineering it should be possible to generate short peptides with selective antibacterial activity.     

Towards a structure-function analysis of bovine lactoferricin and related tryptophan- and arginine-containing peptides.

The iron-binding protein lactoferrin is a multifunctional protein that has antibacterial, antifungal, antiviral, antitumour, anti-inflammatory, and immunoregulatory properties. All of these additional properties appear to be related to its highly basic N-terminal region. This part of the protein can be released in the stomach by pepsin cleavage at acid pH. The 25-residue antimicrobial peptide that is released is called lactoferricin. In this work, we review our knowledge about the structure of the peptide and attempt to relate this to its many functions. Microcalorimetry and fluorescence spectroscopy data regarding the interaction of the peptide with model membranes show that binding to net negatively charged bacterial and cancer cell membranes is preferred over neutral eukaryotic membranes. Binding of the peptide destabilizes the regular membrane bilayer structure. Residues that are of particular importance for the activity of lactoferricin are tryptophan and arginine. These two amino acids are also prevalent in "penetratins", which are regions of proteins or synthetic peptides that can spontaneously cross membranes and in short hexapeptide antimicrobial peptides derived through combinatorial chemistry. While the antimicrobial, antifungal, antitumour, and antiviral properties of lactoferricin can be related to the Trp/Arg-rich portion of the peptide, we suggest that the anti-inflammatory and immunomodulating properties are more related to a positively charged region of the molecule, which, like the alpha- and beta-defensins, may act as a chemokine. Few small peptides are involved in as wide a range of host defense functions as bovine and human lactoferricin.     

Translocating proline-rich peptides from the antimicrobial peptide bactenecin 7

The intracellular delivery of most peptides, proteins, and nucleotides to the cytoplasm and nucleus is impeded by the cell membrane. To allow simplified, noninvasive delivery of attached cargo, cell-permeant peptides that are either highly cationic or hydrophobic have been utilized. Because cell-permeable peptides share half of the structural features of antimicrobial peptides containing clusters of charge and hydrophobic residues, we have explored antimicrobial peptides as templates for designing cell-permeant peptides. We prepared synthetic fragments of Bac 7, an antimicrobial peptide with four 14-residue repeats from the bactenecin family. The dual functions of cell permeability and antimicrobial activity of Bac 7 were colocalized at the N-terminal 24 residues of Bac 7. In general, long fragments of Bac(1-24) containing both regions were bactericidal and cell-permeable, whereas short fragments with only a cationic or hydrophobic region were cell-permeant without the attendant microbicidal activity when measured in a fluorescence quantitation assay and by confocal microscopy. In addition, the highly cationic fragments were capable of traversing the cell membrane and residing within the nucleus. A common characteristic shared by the cell-permeant Bac(1-24) fragments, irrespective of their number of charged cationic amino acids, is their high proline content. A 10-residue proline-rich peptide with two arginine residues was capable of delivering a noncovalently linked protein into cells. Thus, the proline-rich peptides represent a potentially new class of cell-permeant peptides for intracellular delivery of protein cargo. Furthermore, our results suggest that antimicrobial peptides may represent a rich source of templates for designing cell-permeant peptides.     

Antibacterial activity of 15-residue lactoferricin derivatives.

Lactoferricins are a class of antibacterial peptides isolated after gastric-pepsin digest of the mammalian iron-chelating-protein lactoferrin. For investigation of antibacterial activity, we prepared short synthetic derivatives of bovine, human, caprine, murine and porcine lactoferricins with 15-amino-acid residues of high sequence homology. The peptides corresponded to amino-acid residues 17-31 of the mature bovine lactoferrin. Only the bovine and caprine derivatives displayed measurable antibacterial activity, with the bovine one having a minimal inhibitory concentration of 24 microM and being 10 times more active than the caprine one against Escherichia coli. An alanine-scan of the bovine lactoferricin derivative was performed to identify specific amino acids that were important for the antibacterial activity. We found that neither of the two tryptophan residues (Trp 6 and Trp 8) present in the bovine lactoferricin derivative could be replaced by alanine without a major loss of antibacterial activity. The other lactoferricin derivatives tested contained only one tryptophan residue (Trp 6). Modified human, caprine and porcine lactoferricin derivatives containing two tryptophan residues (Trp 6 and Trp 8) displayed minimal inhibitory concentrations of 74, 174 and 219 microM, respectively, which represented up to a six-fold increase in antibacterial activity. The alanine-scan also revealed that the antibacterial activity was increased when acetamidomethyl-protected cysteine and unprotected glutamine (Cys 3 and Gln 7) were replaced with alanine. Only the bovine lactoferricin derivative and a few of its alanine-modified derivatives displayed measurable activity against Staphylococcus aureus.