Isolation and characterization of four antibacterial peptides from bovine hemoglobin

Peptic digestion of bovine hemoglobin at low degree of hydrolysis yields several intermediate peptide fractions after separation by reversed phase HPLC exhibiting antibacterial activity against Micrococcus luteus A270, Listeria innocua, Escherichia coli, and Salmonella enteritidis. From these fractions, four new antibacterial peptides were isolated and analyzed by ESI-MS/MS. Three of these peptides correspond to fragments of the alpha-chain of bovine hemoglobin: alpha107-141, alpha137-141, and alpha133-141, and one peptide to the beta-chain: beta126-145. The minimum inhibitory concentrations (MIC) of these peptides towards the four strains and their hemolytic activity towards bovine erythrocytes were determined.     

Design of potent, non-toxic antimicrobial agents based upon the structure of the frog skin peptide, pseudin-2

Pseudin-2, a naturally occurring 24 amino-acid-residue antimicrobial peptide first isolated from the skin of the South American paradoxical frog Pseudis paradoxa, has weak hemolytic and cytolytic activity but also relatively low potency against microorganisms. In a membrane-mimetic environment, the peptide exists in an amphipathic alpha-helical conformation. Analogs of the peptide with increased cationicity and alpha-helicity were chemically synthesized by progressively substituting neutral and acidic amino acid residues on the hydrophilic face of the alpha-helix by lysine. Analogs with up to three L-lysine substitutions showed increased potency against a range of gram-negative and gram-positive bacteria (up to 16-fold) whilst retaining low hemolytic activity. The analog [D-Lys3, D-Lys10, D-Lys14]pseudin-2 showed potent activity against gram-negative bacteria (minimum inhibitory concentration, MIC=5 microM against several antibiotic-resistant strains of Escherichia coli) but very low hemolytic activity (HC50>500 microM) and cytolytic activity against L929 fibroblasts (LC50=215 microM). Increasing the number of l-lysines to four and five did not enhance antimicrobial potency further but increased hemolytic activity towards human erythrocytes. Time-kill studies demonstrated that the analog [Lys3, Lys10, Lys14, Lys21]pseudin-2 at a concentration of 1 x MIC was bacteriocidal against E. coli (99.9% cell death after 96 min) but was bacteriostatic against S. aureus. Increasing the hydrophobicity of pseudin-2, while maintaining the amphipathic character of the molecule, by substitution of neutral amino acids on the hydrophobic face of the alpha-helix by L-phenylalanine, had only minor effects on antimicrobial and hemolytic activities.     

Isolation and characterization of an antimicrobial peptide from bovine hemoglobin ��-subunit

In this study, a novel 18-residue linear antimicrobial peptide derived from the central part of the bovine hemoglobin ??-subunit was identified. The peptide was purified by a combination of cationic exchange and reversed-phase high-performance liquid chromatography. The sequence was determined to be VNFKLLSHSLLVTLASHL. The theoretical molecular weight of this peptide was calculated to be 1992.38 Da, which is the same as that determined (1992.401 Da) by matrix-assisted laser desorption ionization mass spectrometry. Sequence analysis showed that there is a high degree of homology in this peptide among hemoglobin ??-subunits of bovine, sheep, deer, porcine, and human. In a radial-diffusion plate assay, this purified peptide exhibited antimicrobial activity against Escherichia coli, Staphylococcus aureus, and Candida albicans.     

Identification of two distinct antibacterial domains within the sequence of bovine alpha(s2)-casein.

Two distinct domains with antibacterial activity were isolated from a peptic hydrolysate of bovine alpha(s2)-casein. The digested alpha(s2)-casein was fractionated by cation-exchange chromatography, after which the peptides in the two active fractions obtained were separated by high-performance liquid chromatography and sequenced by electrospray-ionization tandem mass spectrometry. The major component in each active fraction, f(183-207) and f(164-179), was further purified and the antibacterial activity of these components was tested against several microorganisms. Depending on the target bacterial strain, these peptides exhibited minimum inhibitory concentrations between 8 and 99 microM. Peptide f(183-207) exhibited a consistently higher antibacterial activity than f(164-179), although both peptides showed a comparable hemolytic effect. A method of in situ enzymatic hydrolysis on a cation-exchange membrane to obtain a fraction enriched in the most active antibacterial domain is presented. The antibacterial and hemolytic activities are discussed in relation to the structure and hydrophobicity of the peptides.     

Antimicrobial activity of a bovine hemoglobin fragment in the tick Boophilus microplus.

Antifungal and antibacterial activities were detected in the hemolymph and gut contents of the cattle tick, Boophilus microplus. A peptide with antibacterial activity from the tick gut contents was purified to homogeneity by reversed-phase chromatography. The molecular mass of the purified peptide was 3,205.7 Da, measured by matrix-assisted laser desorption/ionization mass spectrometry. The amino acid sequence was obtained by Edman degradation and showed that the peptide was identical to a fragment of the bovine alpha-hemoglobin. A synthetic peptide based on the sequence obtained showed characterization data identical to those of the isolated material, confirming its structure. The synthetic peptide was active in micromolar concentrations against Gram-positive bacteria and fungi. These data led us to conclude that the antibacterial activity detected in tick gut contents is the result of enzymatic processing of a host protein, hemoglobin. This activity may be used by ticks as a defense against microorganisms.     

Isolation and characterization of angiotensin I-converting enzyme inhibitory peptides derived from porcine hemoglobin

Animal blood is potentially an untapped source of drugs and value-added food production. More than 400 million pigs are slaughtered each year but porcine blood is usually discarded in China. This study describes the isolation and characterization of angiotensin I-converting enzyme (ACE) inhibitory peptides derived from porcine hemoglobin. The most active hydrolysate was obtained from the peptic digestion of porcine hemoglobin. After the purification of ACE-inhibitory peptides with Sephadex LH-20 gel chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC) on C(18) column, two active fractions were obtained. They were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). They were LGFPTTKTYFPHF and VVYPWT, corresponding to the 34-46 fragment of the alpha chain and the 34-39 fragment of the beta chain of porcine hemoglobin, with IC(50) values of 4.92 and 6.02 microM, respectively. They were the first found from porcine hemoglobin; in particular, LGFPTTKTYFPHF was a novel ACE-inhibitory peptide. In addition, the purified ACE inhibitors both competitively inhibited ACE, and maintained inhibitory activity even after incubation with gastrointestinal proteases. This suggests that these peptides might have a potential antihypertensive effect.     

Minimal antimicrobial peptidic sequence from hemoglobin alpha-chain: KYR

Hemoglobin is an animal protein described as a source of biologically active peptides. Peptic digestion of bovine hemoglobin alpha-chain allowed obtaining peptide fractions with antimicrobial activity. These peptides were purified by reverse-phase High-Performance Liquid Chromatography (HPLC) and characterized by mass spectrometry. The minimal inhibitory concentration and mode of action of these peptides were studied against five bacterial strains including Escherichia coli and Salmonella enteritidis as Gram-negative bacteria and Listeria innocua, Micrococcus luteus and Staphylococcus aureus as Gram-positive bacteria. The action aforementioned peptides were studied on artificial membranes as well. The most active peptides resulted to be the short ones. Consequently, the minimal peptidic sequence necessary for the antibacterial activity was clearly determined: KYR.     

Activities of the frog skin peptide, ascaphin-8 and its lysine-substituted analogs against clinical isolates of extended-spectrum beta-lactamase (ESBL) producing bacteria

Extended-spectrum beta-lactamase (ESBL)-producing Gram-negative bacteria are becoming increasingly prevalent and their antibiotic resistance necessitates novel therapeutic intervention. Ascaphin-8 is a cationic alpha-helical peptide that shows broad-spectrum antibacterial activity but is also toxic to human erythrocytes (LC(50)= 55 microM). This study assesses the activity of ascaphin-8, and a series of l-lysine-substituted analogs, against a range of clinical isolates of ESBL-producing bacteria. All ESBL-producing Escherichia coli (MIC=1.5-6 microM) and Klebsiella pneumoniae (MIC=12.5-25 microM) strains tested were susceptible to ascaphin-8, as well as a group of miscellaneous ESBL-producing strains (Citrobacter, Salmonella, Serratia, Shigella spp.) (MIC< or = 25 microM). The Lys4- and Lys8-substituted analogs were generally the most potent against bacteria but showed the highest hemolytic activity. However, the Lys10, Lys14, and Lys18 analogs also displayed potent antibacterial activity while showing very low hemolytic activity (LC50> 500 microM). An unexpected finding was the susceptibility of ESBL-producing Proteus mirabilis strains to ascaphin-8 (MIC=12.5-25 microM) and its Lys4-substituted analog (MIC= 6 microM), although non-ESBL isolates of this organism were resistant to these peptides (MIC> 100 microM).     

Sub-femtomole peptide detection in ion mobility-time-of-flight mass spectrometry measurements

Sensitivity and detection limit enhancements are obtained for peptides by performing high repetition rate (150 Hz) matrix-assisted laser desorption/ionization (MALDI) coupled with ion mobility-time-of-flight mass spectrometry. Absolute limits of detection (3sigma) for model peptides are on the order of 0.1 fmol of peptide deposited and represent a factor of 40-60 improvement over data obtained using typical low repetition (20 Hz) MALDI. This increase in sensitivity is demonstrated for two-dimensional MALDI-IM-TOFMS peptide mass mapping of bovine hemoglobin.     

Isolation and characterization of a bacterial growth-stimulating peptide from a peptic bovine hemoglobin hydrolysate

 A peptide with a bacterial-growth-stimulating activity was isolated from a bovine hemoglobin hydrolysate by reversed-phase high-performance liquid chromatography. Its primary structure and molecular mass, determined by amino acid analysis and fast-atom bombardment mass spectrometry, were identical to those of fragment 48–52 (Ser-Thr-Ala-Asp-Ala) of the β chain of bovine hemoglobin. The microbiological tests in solid media demonstrated that this peptide exhibited a growth-stimulating activity on gram-negative bacteria. Received: 18 September 1995/Received revision: 19 January 1996/Accepted: 29 January 1996     

Antimicrobial peptides from diverse families isolated from the skin of the Asian frog, Rana grahami

Seven peptides with antimicrobial activity were isolated in pure form from an extract of the skin of the Yunnanfu Kunming frog Rana grahami Boulenger, 1917. The peptides were identified as belonging to the nigrocin-2 (three peptides), brevinin-1 (one peptide), brevinin-2 (three peptides), and esculentin-1 (one peptide) families. Nigrocin-2GRb (GLFGKILGVGKKVLCGLSGMC) containing three lysine residues, represented the peptide with highest potency against microorganisms (MIC = 3 microM against Escherichia coli, 12.5 microM against Staphylococcus aureus and 50 microM against Candida albicans) and the greatest hemolytic activity against human erythrocytes (LD50 = 40 microM). In contrast, nigrocin-2GRa (GLLSGILGAGKHIVCGLSGLC) and nigrocin-2GRc (GLLSGILGAGKNIVCGLSGLC), with only a single lysine residue, showed weak antimicrobial and hemolytic activity. Phylogenetic relationships among Eurasian ranid frogs are less well understood than those of North American ranids but the primary structures of the R. grahami antimicrobial peptides suggest a close relationship of this species with the Japanese pond frogs R. nigromaculata and R. porosa brevipoda.     

Hipposin,a histone-derived antimicrobial peptide in Atlantic halibut (Hippoglossus hippoglossus L.)

A novel 51-residue antimicrobial peptide (AMP) from the skin mucus of Atlantic halibut (Hippoglossus hippoglossus L.) was isolated using acid extraction, and cationic exchange and reversed phase chromatography. The complete amino acid sequence of the AMP, termed hipposin, was determined by automated Edman degradation and mass spectrometry to be SGRGKTGGKARAKAKTRSSRAGLQFPVGRVHRLLRKGNYAHRVGAGAPVYL. The N-terminal amino group was acetylated. The theoretical mass of hipposin was calculated to be 5458.4 Da, which was in good agreement with the mass of 5459 Da determined by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Hipposin was shown to be derived from histone H2A by PCR amplifying the encoding sequences from Atlantic halibut genomic DNA. The peptide showed sequence similarity with the 39-mer AMP buforin I of Asian toad and the 19-mer AMP parasin I of catfish. Fifty of the fifty-one residues in hipposin were identical to the N-terminal region of histone H2A from rainbow trout. Hipposin showed strong antimicrobial activity against several Gram-positive and Gram-negative bacteria and activity could be detected down to hipposin concentrations of 0.3 microM (1.6 microg/ml). Hipposin without N-terminal acetylation was prepared by solid-phase peptide synthesis and shown to have the same antimicrobial activity as the natural acetylated peptide. Thus, hipposin is a new broad-spectrum histone-derived AMP found in the skin mucus of Atlantic halibut.     

Identification of an antihypertensive peptide from peptic digest of wakame (Undaria pinnatifida)

A peptide fraction having activity against angiotensin I-converting enzyme (ACE) was separated from the peptic digest of protein prepared from wakame (Undaria pinnatifida) by ion-exchange chromatographies and gel-filtration. Fractions with high ACE inhibitory activity were combined and further chromatographed on a reverse-phase column to yield four tetrapeptides with ACE inhibitory properties. These tetrapeptides were identified by sequence analysis and fast atom bombardment mass spectrometry as Ala-Ile-Tyr-Lys (IC(50): 213 microM), Tyr-Lys-Tyr-Tyr (64.2 microM), Lys-Phe-Tyr-Gly (90.5 microM), and Tyr-Asn-Lys-Leu (21 microM). Each tetrapeptide was synthesized and its antihypertensive activity was determined after oral administration in spontaneously hypertensive rats. The blood pressure significantly decreased after tetrapeptide ingestion. The present study demonstrated that dietary wakame may have beneficial effects on hypertension.     

Purification and antimicrobial activity studies of the N-terminal fragment of ubiquitin from human amniotic fluid

A 4.3-kDa antimicrobial peptide was isolated from human amniotic fluid by dialysis, ultrafiltration, and C18 reversed-phase high performance liquid chromatography. This peptide, which we named Amniotic Fluid Peptide-1 (AFP-1), possessed antimicrobial activity but lacked hemolytic activity. In addition, AFP-1 potently inhibited the growth of a variety of bacteria (Escherichia coli, Salmonella typhimurium, Listeria monocytogenes and Staphylococcus aureus), filamentous fungi (Botrytis cinerea, Aspergillus fumigatus, Neurospora crassa and Fusarium oxysporum) and yeast cells (Candida albicans and Cryptococcus neoformans). Automated Edman degradation showed that the N-terminal sequence of AFP-1 was NH(2)-Met-Gln-Ile-Phe-Val-Lys-Thr-Leu-Thr-Gly-Lys-Thr-Ile-Thr-Leu-Glu-Val-Glu-. The partial sequence had 100% homology to the N-terminal sequence of ubiquitin. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry revealed that the molecular mass of AFP-1 was 4280.2 Da. Our data show an antimicrobial activity of ubiquitin N-terminal derived peptide that makes it suitable for use as an antimicrobial agent.     

De novo designed cyclic cationic peptides as inhibitors of plant pathogenic bacteria

Head-to-tail cyclic peptides of 4-10 residues consisting of alternating hydrophilic (Lys) and hydrophobic (Leu and Phe) amino acids were synthesized and tested against the economically important plant pathogenic bacteria Erwinia amylovora, Xanthomonas vesicatoria and Pseudomonas syringae. The antibacterial activity, evaluated as the minimal inhibitory concentration (MIC), the cytotoxicity against human red blood cells and stability towards protease degradation were determined. The influence of cyclization, ring size, and replacement of l-Phe with d-Phe on antibacterial and hemolytic activities was studied and correlated with the degree of structuring and hydrophobicity. Our results showed that linear peptides were inactive against the three bacteria tested. Cyclic peptides were active only toward X. vesicatoria and P. syringae, being c(KLKLKFKLKQ) (BPC10L) the most active peptide with MIC values of 6.25 and 12.5 microM, respectively. The improved antibacterial activity of cyclic peptides compared to their linear counterparts was associated to an increase of the hydrophobicity, represented as RP-HPLC retention time (t(R)), and secondary structure content which are related to an enhanced amphipathicity. A decrease of antibacterial and hemolytic activities was observed when a d-Phe was introduced into the cyclic sequences, which was attributed to their low amphipathicity as shown by their low secondary structure content and low t(R). The small size, simple structure, bactericidal effect, and stability to protease degradation of the best peptides make them potential candidates for the development of effective antibacterial agents for use in plant protection.     

Antimicrobial and cytolytic properties of the frog skin peptide, kassinatuerin-1 and its L- and D-lysine-substituted derivatives

Kassinatuerin-1, a 21-amino-acid C-terminally alpha-amidated peptide first isolated from the skin of the African frog Kassina senegalensis, adopts an amphipathic alpha-helical conformation in a membrane-mimetic solvent (50% trifluoroethanol) and shows broad-spectrum antimicrobial activity. However, its therapeutic potential is limited by its relatively high cytolytic activity against mammalian cells. The antimicrobial and cytolytic properties of a peptide are determined by an interaction between cationicity, hydrophobicity, alpha-helicity and amphipathicity. Replacement of the C-terminal alpha-amide group in kassinatuerin-1 by carboxylic acid decreased both cationicity and alpha-helicity, resulting in an analog with decreased potency against Escherichia coli (4-fold) and Staphylococcus aureus (16-fold). Low cytolytic activities against human erythrocytes (LD50>400 microM) and L929 fibroblasts (LD50=105 microM) were also observed. Increasing cationicity, while maintaining amphipathic alpha-helical character, by progressively substituting Gly7, Ser18, and Asp19 on the hydrophilic face of the alpha-helix with L-lysine, increased antimicrobial potency against S. aureus and Candida albicans (up to 4-fold) but also increased hemolytic and cytolytic activities. In contrast, analogs with d-lysine at positions 7, 18 and 19 retained activity against Gram-negative bacteria but displayed reduced hemolytic and cytolytic activities. For example, the carboxylic acid derivative of [D-Lys7, D-Lys18, D-Lys19]kassinatuerin-1 was active (minimum inhibitory concentration (MIC)=6-12.5 microM) against a range of strongly antibiotic-resistant strains of E. coli but showed no detectable hemolytic activity at 400 microM and was 4-fold less cytolyic than kassinatuerin-1. However, the reduction in alpha-helicity produced by the D-amino acid substitutions resulted in analogs with reduced potencies against Gram-positive bacteria and against C. albicans.     

A family of brevinin-2 peptides with potent activity against Pseudomonas aeruginosa from the skin of the Hokkaido frog, Rana pirica

Nine peptides displaying varying degrees of antimicrobial activity were extracted from the skin of the Hokkaido frog, Rana pirica. Five structurally related peptides were identified as members of the brevinin-2 family. These peptides were active against reference strains of Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella pneumoniae) and Gram-positive (Staphlococcus aureus) bacteria but displayed relatively low hemolytic activity. The most abundant peptide, brevinin-2PRa (680 nmol/g weight of dry skin) showed high potency [minimal inhibitory concentration (MIC) values between 6 and 12 microM] against a range of clinical isolates of P. aeruginosa. In addition, activity was unaffected by NaCl concentrations up to 200 mM. Cladistic analysis based on the primary structures of brevinin-2 peptides supports a close phylogenetic relationship between R. pirica and Japanese mountain brown frog Rana ornativentris. One peptide of the ranatuerin-2 family and one strongly hemolytic peptide of the brevinin-1 family were also isolated from the extract along with two members of the temporin family, temporin-1PRa (ILPILGNLLNGLL.NH(2)) and temporin-1PRb (ILPILGNLLNSLL.NH(2)) that atypically lacked basic amino acid residues and showed only very weak antimicrobial and hemolytic activity.     

Peptide enrichment by microfluidic electrocapture for online analysis by electrospray mass spectrometry

Identification of peptides from a complex mixture can be difficult because of the wide concentration range and the different ionization efficiencies of peptides during analysis by electrospray ionization (ESI) mass spectrometry (MS). Preconcentration methods are necessary to allow low-abundance and low-intensity peptides to reach the ionization threshold of the mass spectrometer. Here we demonstrate peptide enrichment based on electroimmobilization. Peptides are immobilized without the use of solid support or chemical binding by application of an electric field along a microflow stream in an electrocapture cell. Once enriched/preconcentrated inside the cell, they are released by removal of the electric field and via an interface with an electrospray emitter are submitted to online mass spectrometric analysis. Tandem mass spectrometric analysis of a peptide mixture containing hemoglobin, myoglobin, bovine serum albumin (BSA), and cytochrome c was successful. Amplification factors up to 16-fold were achieved with improvement of the signal-to-noise values for the preconcentrated sample. The limit of detection for one of the preconcentrated peptides was 3.6 fmol.     

Conolysin-Mt: a conus peptide that disrupts cellular membranes

Conus venoms are estimated to comprise over 100,000 distinct pharmacologically active peptides, the majority probably targeting ion channels. Through the characterization of a cytolytic peptide from the venom of Conus mustelinus, conolysin-Mt, we expand the known conopeptide mechanisms to include association with and destruction of cellular membranes. A new 23AA conopeptide, conolysin-Mt has potent hemolytic activity when tested on human erythrocytes. At a concentration of 0.25 microM, the peptide permeabilized both negatively charged prokaryotic (PE:PG) and zwitterionic eukaryotic (PC:cholesterol) model membranes. The affinity constants (KA) of conolysin-Mt for PE:PG and PC:cholesterol model membranes were 0.9 +/- 0.3 x 10(7) and 3 +/- 1 x 10(7) M-1, respectively. In contrast, conolysin-Mt exhibited low antimicrobial activity (MIC > 50 microM) against two Escherichia coli strains, with an MIC for the Gram-positive S. aureus of 25-50 microM. The specificity of conolysin-Mt for native eukaryotic membranes is a novel feature of the peptide compared to other well-characterized cytolytic peptides such as melittin.     

A short α-helical antimicrobial peptide with antibacterial selectivity

A 13-residue alpha-helical peptide (K6L5WP), designed from Leu6-->Pro substitution of a hemolytic alpha-helical peptide (K6L6W), exhibited strong antibacterial activity (MIC: 2 to approximately 4 microM against three gram-positives and three gram-negatives) comparable to that of melittin but had no hemolytic activity. Tryptophan fluorescence studies indicated bacterial selectivity of K6L5WP is closely related to the selective interaction with negatively charged phospholipids on the surface of bacterial cells. These results suggested that the central Pro6 in K6L5WP plays an important role in its bacterial cell selectivity. In conclusion, K6L5WP with antibacterial selectivity may serve as an attractive candidate for the development of antimicrobial agents.