Conformation and activity of delta-lysin and its analogs

Delta-Lysin is a 26-residue hemolytic peptide secreted by Staphylococcus aureus. Unlike the bee venom peptide melittin, delta-lysin does not exhibit antibacterial activity. We have synthesized delta-lysin and several analogs wherein the N-terminal residues of the toxin were sequentially deleted. The toxin has three aspartic acids, four lysines and no prolines. Analogs were also generated in which all the aspartic acids were replaced with lysines. A proline residue was introduced in the native sequences as well as in the analogs where aspartic acids were replaced with lysines. We observed that 20- and 22-residue peptides corresponding to residues 7-26 and 5-26 of delta-lysin, respectively, had greater hemolytic activity than the parent peptide. These shorter peptides, unlike delta-lysin, did not self-associate to adopt alpha-helical conformation in water, at lytic concentrations. Introduction of proline or substitution of aspartic acids by lysines resulted in loss in propensity to adopt helical conformation in water. When proline was introduced in the peptides corresponding to the native toxin sequence, loss of hemolytic activity was observed. Substitution of all the aspartic acids with lysines resulted in enhanced hemolytic activity in all the analogs. However, when both proline and aspartic acid to lysine changes were made, only antibacterial activity was observed in the shorter peptides. Our investigations on delta-lysin and its analogs provide insights into the positioning of anionic, cationic residues and proline in determining hemolytic and antibacterial activities.     

The effect of acidic residues and amphipathicity on the lytic activities of mastoparan peptides studied by fluorescence and CD spectroscopy

Some mastoparan peptides extracted from social wasps display antimicrobial activity and some are hemolytic and cytotoxic. Although the cell specificity of these peptides is complex and poorly understood, it is believed that their net charges and their hydrophobicity contribute to modulate their biological activities. We report a study, using fluorescence and circular dichroism spectroscopies, evaluating the influence of these two parameters on the lytic activities of five mastoparans in zwitterionic and anionic phospholipid vesicles. Four of these peptides, extracted from the venom of the social wasp Polybia paulista, present both acidic and basic residues with net charges ranging from +1 to +3 which were compared to Mastoparan-X with three basic residues and net charge +4. Previous studies revealed that these peptides have moderate-to-strong antibacterial activity against Gram-positive and Gram-negative microorganisms and some of them are hemolytic. Their affinity and lytic activity in zwitterionic vesicles decrease with the net electrical charges and the dose response curves are more cooperative for the less charged peptides. Higher charged peptides display higher affinity and lytic activity in anionic vesicles. The present study shows that the acidic residues play an important role in modulating the peptides’ lytic and biological activities and influence differently when the peptide is hydrophobic or when the acidic residue is in a hydrophilic peptide.     

Venom peptides from solitary hunting wasps induce feeding disorder in lepidopteran larvae

The cell lytic activity and toxicity against lepidopteran larvae of 13 venom peptides (4 OdVPs and 9 EpVPs) from two solitary hunting wasps, Orancistrocerus drewseni and Eumenes pomiformis, were examined with mastoparan as a reference peptide. Of the 13 peptides, 7 were predicted to have α-helical structures that exhibit the typical character of amphipathic α-helical antimicrobial peptides. The remaining peptides exhibited coil structures; among these, EpVP5 possesses two Cys residues that form an internal disulfide bridge. All the helical peptides including mastoparan showed antimicrobial and insect cell lytic activities, whereas only two of them were hemolytic against human erythrocytes. The helical peptides induced a feeding disorder when injected into the vicinity of the head and thorax of Spodoptera exigua larvae, perhaps because their non-specific neurotoxic or myotoxic action induced cell lysis. At low concentrations, however, these helical peptides increased cell permeability without inducing cell lysis. These findings suggest that the helical venom peptides may function as non-specific neurotoxins or myotoxins and venom-spreading factors at low concentrations, as well as preservatives for long-term storage of the prey via antimicrobial, particularly antifungal, activities.     

Isolation and biochemical characterization of peptides presenting antimicrobial activity from the skin of Phyllomedusa hypochondrialis

Amphibian antimicrobial peptides have been known for many decades and several of them have been isolated from anuran species. Dermaseptins are among the most studied antimicrobial peptides and are found in the skin secretion of tree frogs from the Phyllomedusinae subfamily. These peptides exert a lytic action on bacteria, protozoa, yeast, and filamentous fungi at micromolar concentrations, but unlike polylysines, present little hemolytic activity. In this work, two antimicrobial peptides were isolated from the crude skin secretion of Phyllomedusa hypochondrialis and tested against Gram-positive and Gram-negative bacteria, presenting no hemolytic activity at the tested concentrations. One of them was identified with the recently reported peptide PS-7 belonging to the phylloseptin family, and another was a novel peptide, named DPh-1, which was fully purified, sequenced by ‘de novo’ mass spectrometry and grouped into Dermaseptins (DPh-1).     

Solution conformations of peptides representing the sequence of the toxin pardaxin and analogues in trifluoroethanol-water mixtures: Analysis of CD spectra

The cytolytic activities and conformational properties of pardaxin (GFFALIPKIISSPLFKTLLSAVGSALSSSGEQE), a 33-residue linear peptide that exhibits unusual shark repellent and cytolytic activities, and its analogues have been examined in aqueous environment and trifluoroethanol (TFE) using CD spectroscopy. A peptide corresponding to the 1–26 segment and an analogue where P7 has been changed to A show greater hemolytic activity than pardaxin. While the peptide corresponding to the N-terminal 18-residue segment does not exhibit hemolytic activity, its analogue where P7 is replaced by A is hemolytic. The secondary structural propensities of the peptides were inferred by deconvolution of the experimental spectra into pure components. Pardaxin, its variant where proline at position 7 was replaced by alanine, and shorter peptides corresponding to N-terminal segments exist in multiple conformations in aqueous medium that are comprised of β-turn, β-sheet, and distorted helical structures. With increasing proportions of TFE, while helical conformation predominates in all the peptides, both distorted and the regular α-helices appear to be populated. Analysis of CD spectra by deconvolution methods appears to be a powerful tool for delineating multiple conformations in peptides, especially membrane-active peptides that encounter media of different polarity ranging from aqueous environment to one of low dielectric constant in the hydrophobic interior of membranes. Our study provides further insights into the structural requirements for the biological activity of pardaxin and related peptides. © 1997 John Wiley & Sons, Inc. Biopoly 41: 635–645, 1997     

The effect of cyclization of magainin 2 and melittin analogues on structure, function, and model membrane interactions: implication to their mode of action

The amphipathic alpha-helical structure is a common motif found in membrane binding polypeptides including cell lytic peptides, antimicrobial peptides, hormones, and signal sequences. Numerous studies have been undertaken to understand the driving forces for partitioning of amphipathic alpha-helical peptides into membranes, many of them based on the antimicrobial peptide magainin 2 and the non-cell-selective cytolytic peptide melittin, as paradigms. These studies emphasized the role of linearity in their mode of action. Here we synthesized and compared the structure, biological function, and interaction with model membranes of linear and cyclic analogues of these peptides. Cyclization altered the binding of melittin and magainin analogues to phospholipid membranes. However, at similar bound peptide:lipid molar ratios, both linear and cyclic analogues preserved their high potency to permeate membranes. Furthermore, the cyclic analogues preserved approximately 75% of the helical structure of the linear peptides when bound to membranes. Biological activity studies revealed that the cyclic melittin analogue had increased antibacterial activity but decreased hemolytic activity, whereas the cyclic magainin 2 analogue had a marked decrease in both antibacterial and hemolytic activities. The results indicate that the linearity of the peptides is not essential for the disruption of the target phospholipid membrane, but rather provides the means to reach it. In addition, interfering with the coil-helix transition by cyclization, while maintaining the same sequence of hydrophobic and positively charged amino acids, allows a separated evaluation of the hydrophobic and electrostatic contributions to binding of peptides to membranes.     

Melittin peptides exhibit different activity on different cells and model membranes

Melittin (MLT) is a lytic peptide with a broad spectrum of activity against both eukaryotic and prokaryotic cells. To understand the role of proline and the thiol group of cysteine in the cytolytic activity of MLT, native MLT and cysteine-containing analogs were prepared using solid phase peptide synthesis. The antimicrobial and cytolytic activities of the monomeric and dimeric MLT peptides against different cells and model membranes were investigated. The results indicated that the proline residue was necessary for antimicrobial activity and cytotoxicity and its absence significantly reduced lysis of model membranes and hemolysis. Although lytic activity against model membranes decreased for the MLT dimer, hemolytic activity was increased. The native peptide and the MLT-P14C monomer were mainly unstructured in buffer while the dimer adopted a helical conformation. In the presence of neutral and negatively charged vesicles, the helical content of the three peptides was significantly increased. The lytic activity, therefore, is not correlated to the secondary structure of the peptides and, more particularly, on the propensity to adopt helical conformation.     

Effect of multiple aliphatic amino acids substitutions on the structure, function, and mode of action of diastereomeric membrane active peptides

The initial stages leading to the binding and functioning of membrane-active polypeptides including hormones, signal sequences, and lytic peptides are mainly governed by electrostatic attraction and hydrophobic partitioning between water and lipid bilayers. Antimicrobial peptides serve as an important model for studying the details of these initial steps. However, a systematic analysis of the contribution of multiple hydrophobic amino acids to these steps have been hindered by the propensity of many peptides to aggregate and become inactivated in solution. To this end, we synthesized a series of model amphipathic all L-amino acid peptides and their diastereomers with the sequence KX(3)KWX(2)KX(2)K, where X = Gly, Ala, Val, Ile, or Leu. The effect of the aliphatic amino acids on the biological activity, binding, structure, membrane localization, and mode of action of these peptides was investigated. Most of the L-amino acid peptides oligomerized and adopted distinct structures in solution and in a membrane mimetic environment. Among this group only the Leu containing peptide was hemolytic and highly active on most bacteria tested. The Val- and Leu-containing peptides were hemolytic but inactive toward most bacteria tested. In contrast, the diastereomeric peptides were monomeric and unstructured in solution, but they adopted distinct structures upon membrane binding. While hemolytic activity was drastically reduced, the spectrum of antibacterial activity was preserved or increased. Importantly, we found a direct correlation with the diastereomers between hydrophobicity and propensity to form a helical/distorted-helix and activity (induced membrane leakage and antibacterial activity), despite the fact that they contained 30% D-amino acids. Furthermore, efficient increase in membrane permeability can proceed through different mechanisms. Specifically, the Leu-containing diastereomeric peptide micellized vesicles and possibly bacterial membranes while the Ile-containing diastereomeric peptide fused model membranes and irregularly disrupted bacterial membranes.     

Iodination of a tyrosyl residue in staphylococcal alpha-toxin.

Iodination of staphylococcal alpha-toxin by the lactoperoxidase method resulted in the maximal incorporation of about 2.5 atoms of iodine per molecule of alpha-toxin. The iodination primarily involved a single tyrosine residue as shown by analysis of both cyanogen bromide and tryptic peptides. Iodination at a level of 1.2 iodine atoms per alpha-toxin molecule led to a dramatic decrease in the hemolytic and lethal activities, although no decrease in the binding of iodinated toxin to rabbit erythrocytes was observed (Cassidy and Harshman (1976), Biochemistry, the following paper in this issue). Monoiodinated alpha-toxin was found to have 15% of the specific hemolytic activity of native alpha-toxin. Incubation of rabbit erythrocytes with iodinated alpha-toxin led to a significant protection from the hemolytic activity of native alpha-toxin added later. The results show the modification of a single unique tyrosyl residue in alpha-toxin permits the resolution of alpha-toxin's biological activities from its cell binding activity.     

Coelenterolysin: a hemolytic polypeptide associated with the coelenteric fluid of sea anemones

The gastrovascular fluids of the sea anemone Phymactis clematis display strong hemolytic activity, which has basic pH optimum, is thermolabile and is sensitive to proteases. The hemolytic agent from the gastrovascular fluid was partially purified by ammonium sulfate precipitation, chromatography on Dowex-50W cation exchanger and gel filtration on Sephadex G-50. A single peak elutes from the latter with an estimated mol. wt of 18,000. This elution pattern is unaffected if the chromatography is carried out in the presence of 5 M urea. These results indicate that the hemolytic activity is due to a single peptide or a group of peptides of similar size, which we here designate as “coelenterolysin”. Coelenterolysin is also present in sea anemone tissue homogenates, is different from the nematocyst toxin and is not associated with phospholipase activities. It is inhibited by sphingomyelin. This is the first report of hemolytic polypeptides associated with the coelenteric fluid of sea anemones. Coelenterolysin may have a role in extracellular digestion, defense against predators and invasion of the coelenteron by foreign organisms.     

Synthesis, characterization, and thermodynamic study of a polyvalent lytic peptide-polymer conjugate as novel anticancer agent

We designed and synthesized a new polyvalent lytic peptide-polymer conjugate as a novel chemotherapeutic agent capable of overcoming multidrug resistance. A hexapeptide (KWKWKW or (KW)?) was designed and conjugated to dextran in multiple copies to afford a polyvalent conjugate. A robust synthesis procedure involving click chemistry and the detailed characterization of the conjugate were reported here. The conjugate Dex-(KW)? exhibited significantly enhanced anticancer potency in vitro by up to 500-fold compared to monomeric (KW)?. The LC?? value was comparable to that of conventional lytic peptides which have more than 20 residues. No hemolytic activity was shown by the conjugates up to 300 μM. Thermodynamic study indicated that the binding of conjugates was predominantly entropy-driven while the binding of free peptides was mainly enthalpy-driven, implying a deeper penetration of conjugate into the core of lipid bilayer. The binding affinity of conjugate to neutral membrane is much higher than that to free peptide (K(conj) ≈ 8822.9 M?1, K(pep) ≈ 1884.7 M?1). In binding to negatively charged membrane, the conjugate surpassed free peptides at high concentrations when the binding of free peptides became saturated. The higher binding capability, attributed to the high local concentration of peptides mounted on a polymer backbone, explains the superior anticancer activity of polyvalent Dex-(KW)?.     

Influence of tryptophan on lipid binding of linear amphipathic cationic antimicrobial peptides

We recently demonstrated that a linear 18-residue peptide, (KIGAKI)(3)-NH(2), designed to form amphipathic beta-sheet structure when bound to lipid bilayers, possessed potent antimicrobial activity and low hemolytic activity. The ability of (KIGAKI)(3)-NH(2) to induce leakage from lipid vesicles was compared to that of the amphipathic alpha-helical peptide, (KIAGKIA)(3)-NH(2), which had equivalent antimicrobial activity. Significantly, the lytic properties of (KIGAKI)(3)-NH(2) were enhanced for mixed acidic-neutral lipid vesicles containing phosphatidylethanolamine instead of phosphatidylcholine as the neutral component, while the potency of (KIAGKIA)(3)-NH(2) was significantly reduced [Blazyk, J., et al. (2001) J. Biol. Chem. 276, 27899-27906]. In this paper, we measured the lytic properties of these peptides, as well as several fluorescent analogues containing a single tryptophan residue, by monitoring permeability changes in large unilamellar vesicles with varying lipid compositions and in Escherichia coli cells. The binding of these peptides to lipid bilayers with defined compositions was compared using surface plasmon resonance, circular dichroism, and fluorescence spectroscopy. Surprisingly large differences were observed in membrane binding properties, particularly in the case of KIGAKIKWGAKIKIGAKI-NH(2). Since all of these peptides possess the same charge and very similar mean hydrophobicities, the binding data cannot be explained merely in terms of electrostatic and/or hydrophobic interactions. In light of their equivalent antimicrobial and hemolytic potencies, some of these peptides may employ mechanisms beyond simply increasing plasma membrane permeability to exert their lethal effects.     

The hemolytic activity of six arachnid cationic peptides is affected by the phosphatidylcholine-to-sphingomyelin ratio in lipid bilayers

The hemolytic activity of six cationic amphipathic peptides (Oxki1, Oxki2, Pin1, Pin2, IsCT1 and IsCT2) from arachnids strongly depends on the source of red blood cells. The hemolytic activity of the amphipathic peptides was correlated to the phosphocholine-to-sphingomyelin ratio (PC/SM) content, the potency order of which on mammal erythrocytes ranked as follows Guinea pig>pig>sheep. The spider peptides, Oxki1 and Oxki2, prefer small unilamellar vesicles (SUV) composed of PC, but they could not disrupt SUVs made of SM only. Moreover, the membrane-disrupting activity of the scorpion peptide Pin1 was affected by increasing concentrations of SM. Only the scorpion hemolytic peptide Pin2 was able to disrupt SUVs composed merely of SM at high concentrations. Finally, the short scorpion peptides IsCT1 and IsCT2 seem to tolerate high concentrations of SM in the presence of PC for disruption of SUVs; however, the disrupting activities of IsCT1 and IsCT2 are much lower than that of the other four hemolytic peptides. The hemolytic activity caused by all six cationic peptides in mammalian erythrocytes was positively correlated to increases in temperature and increases in the concentration of benzyl alcohol, a membrane fluidizing agent. It was concluded that the hemolytic activity of the cationic peptides strongly depends on the PC/SM content of mammalian erythrocytes, in which cell membranes with a low PC/SM ratio (i.e., of low fluidity) were less disturbed than membranes with a high PC/SM ratio (i.e., of high fluidity).     

Relationship between the tertiary structures of mastoparan B and its analogs and their lytic activities studied by NMR spectroscopy.

Mastoparan B (MP-B), an antimicrobial cationic tetradecapeptide amide isolated from the venom of the hornet Vespa basalis, is an amphiphilic alpha-helical peptide. MP-B possesses a variety of biological activities, such as mast cells degradation histamine release, erythrocyte lysis and inhibition of the growth of gram-positive and gram-negative bacteria. In order to study the relationship between the structure and the biological activity of MP-B, we used four analogs by replacing amino acids with alanine. Tertiary structures of MP-B and its analogs in 2,2,2-trifluoroethanol (TFE)-containing aqueous solution have been determined by NMR spectroscopy and molecular modeling. The results indicate that [Ala4]MP-B and [Ala12]MP-B with higher hydrophobicity adopt a higher content of amphiphilic helical structures, and have better antimicrobial and hemolytic activities than MP-B. However, [Ala3]MP-B and [Ala9]MP-B with lower hydrophobicity have disordered structures. [Ala3]MP-B and [Ala9]MP-B have low antimicrobial activity and much less hemolytic activity relative to MP-B. It is likely that tryptophan residue in MP-B and appropriate hydrophobicity of MP-B to induce alpha-helical structure is essential for the antibacterial and hemolytic activity of MP-B. This study can aid understanding of the structure-activity relationship of MP-B and to design peptides to possess lytic activity.     

Mapping and analysis of the lytic and fusogenic domains of surfactant protein B

Surfactant protein B (SP-B) is a hydrophobic, 79 amino acid peptide that regulates the structure and function of surfactant phospholipid membranes in the airspaces of the lung. Addition of SP-B to liposomes composed of DPPC/PG (7:3) leads to membrane binding, destabilization, and fusion, ultimately resulting in rearrangement of membrane structure. The goal of this study was to map the fusogenic and lytic domains of SP-B and assess the effects of altered fusion and lysis on surface activity. Synthetic peptides were generated to predicted helices and/or interhelical loops of SP-B and tested for fusion, lytic, and surface activities. The N-terminal half of SP-B (residues 1-37), which includes the nonhelical N-terminal amino acids in addition to helices 1 and 2, promoted rapid liposome fusion whereas shorter peptides were significantly less effective. The requirements for optimal surface tension reduction were similar to those for fusion; in contrast, helix 1 (residues 7-22) alone was sufficient for liposome lysis. The C-terminal half of SP-B (residues 43-79), which includes helices 3, 4, and 5, exhibited significantly lower levels of fusogenic, lytic, and surface tension reducing activities compared to the N-terminal region. These results indicate that SP-B fusion, lytic and surface activities map predominantly to the N-terminal half of SP-B. Amino acid substitutions in synthetic peptides corresponding to the N-terminal half of SP-B indicated that, in general, decreased fusion or lytic activities were associated with altered surface tension reducing properties of the peptide. However, the presence of fusion and lytic activities alone could not account for the surface tension reducing property of SP-B. We propose a model in which association of helix 1 with lipids leads to membrane permeabilization but not aggregation; helix 2 mediates membrane cross-linking (aggregation), which, in turn, facilitates lipid mixing, membrane fusion, and interfacial adsorption/surface tension reduction.     

Synthesis and evaluation of redox-sensitive gonadotropin-releasing hormone receptor-targeting peptide conjugates

Lytic peptides have been demonstrated to exhibit obvious advantages in cancer therapy with binding ability toward tumor cells via electrostatic attractions, which are lack of active targeting and aggregation to tumor tissue. In the present study, five conjugated lytic peptides were redesigned and constructed to target gonadotropin releasing hormone receptors (GnRHr), meanwhile, the disulfide bridge was introduced to achieve redox sensitive delivery based on the experience from the preliminary work of lytic peptides P3 and P7. YX-1, was considered to be the most promising for in-depth study. YX-1 possessed high potency (IC₅₀ = 3.16 ± 0.3 μM), low hemolytic effect, and cell membrane permeability in human A2780 ovarian cancer cells. Moreover, YX-1 had prominent pro-apoptotic activity by activating the mitochondria–cytochrome c–caspase apoptotic pathway. The study yielded the conjugate YX-1 with superior properties for antineoplastic activity, which makes it a promising potential candidate for targeting cancer therapy.     

Zymogram and Preliminary Characterization of Lactobacillus helveticus Autolysins

The autolysins of Lactobacillus helveticus ISLC5 were detected and partially characterized by renaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with substrate-containing gels (zymogram). By using lyophilized Micrococcus luteus cells or heated whole cells of L. helveticus ISLC5 (0.2% [wt/vol]) as a substrate, several lytic activities were detected in the whole-cell SDS extract of strain ISLC5 (i) one activity at 42.4 kDa, which was named autolysin A, and (ii) six other activities having very similar molecular weights (29.1, 29.6, 30, 30.8, 31.7, and 32.8 kDa), which were named autolysins B (B1 through B6, respectively). As regards the temporal distribution of the enzymes, autolysins A and B were detected in the cells harvested from the beginning of the exponential growth phase. Autolysin A appeared to be associated only with viable cells, whereas the autolysins B remained associated with the cell envelope several days after the complete loss of culture viability. When SDS-treated walls of L. helveticus ISLC5 were used as a substrate, a supplementary lytic activity appeared at 37.5 kDa; it was considered a peptidoglycan hydrolase, since it was not able to induce lysis of whole-cell substrate. The autolysins of 30 other strains of L. helveticus from various geographical origins were also analyzed by zymogram; all the activity profiles obtained were similar to that of strain ISLC5 in terms of the number of lytic bands and their apparent molecular weights. Only the relative intensities of the lytic bands corresponding to autolysins A and B were variable depending on the strains. This observation suggested that autolysins are highly conserved enzymes. A concentrated crude lysate of the virulent bacteriophage 832-B1 infecting L. helveticus was also analyzed by zymogram; one lytic activity with an apparent molecular weight of 31.7 kDa, very close to the weights of the autolysins B, was observed. Finally, the autolysins of L. helveticus ISLC5 were successfully extracted from whole cells by using a 1 M lithium chloride solution; they were partially purified by precipitation, selective resolubilization, and gel filtration chromatography, which led to a 20-fold increase in specific activity.     

Biological activities of retro and diastereo analogs of a 13-residue peptide with antimicrobial and hemolytic activities.

The biological activities of synthetic retro and diastereo analogs of PKLLKTFLSKWIG (SPFK), a 13-residue peptide with antimicrobial and hemolytic activities, have been investigated. Retro peptides with C-terminal acid and amide exhibited antibacterial activities comparable with those of SPFK. Their hemolytic activities were, however, only marginally lower. The diastereo analog with C-terminal acid was not antibacterial and was weakly hemolytic. Amidation of this analog could restore antibacterial activity. Both retro analogs were unordered in aqueous medium but had a propensity for a helical structure in trifluoroethanol. However, diastereo analogs were unordered in both aqueous medium and trifluoroethanol. Thus, reversing the sequence in a short amphiphilic peptide may not always result in the selective loss of biological activity such as hemolytic activity. Also, introduction of enantiomeric amino acids in a short peptide to generate a diastereomer may result in loss of structure as well as antimicrobial and hemolytic activities, unless compensated by an increase in positive charges.     

A critical comparison of the hemolytic and fungicidal activities of cationic antimicrobial peptides.

The hemolytic and fungicidal activity of a number of cationic antimicrobial peptides was investigated. Histatins and magainins were inactive against human erythrocytes and Candida albicans cells in phosphate buffered saline, but displayed strong activity against both cell types when tested in 1 mM potassium phosphate buffer supplemented with 287 mM glucose. The HC50/IC50 ratio, indicative of the therapeutic index, was about 30 for all peptides tested. PGLa was most hemolytic (HC50 = 0.6 microM) and had the lowest therapeutic index (HC50/IC50 = 0.5). Susceptibility to hemolysis was shown to increase with storage duration of the erythrocytes and also significant differences were found between blood collected from different individuals. In this report, a sensitive assay is proposed for the testing of the hemolytic activities of cationic peptides. This assay detects subtle differences between peptides and allows the comparison between the hemolytic and fungicidal potency of cationic peptides.