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New antibacterial peptide derived from bovine hemoglobin
Peptides 26 (2005) 713–719
New antibacterial peptide derived from bovine hemoglobin Rachid Daoud a , Veronique Dubois a , Loredana Bors-Dodita a , Naima Nedjar-Arroume a,∗ , Francois Krier a , Nour-Eddine Chihib a , Patrice Mary a , Mostafa Kouach b , Gilbert Briand b , Didier Guillochon a b
a Laboratoire de Technologie des Substances Naturelles, IUT “A” Lille I, BP 179, 59653 Villeneuve d’Ascq Cedex, France Laboratoire d’Application de Spectrom´etrie de Masse, Service Commun de Physicochimie, Facult´e de M´edecine H. Warembourg, Pˆole Recherche, Place de Verdun, Lille II, France
Received 20 October 2004; received in revised form 10 December 2004; accepted 14 December 2004 Available online 19 March 2005
Abstract Peptic digestion of bovine hemoglobin at low degree of hydrolysis yields an intermediate peptide fraction exhibiting antibacterial activity against Micrococcus luteus A270, Listeria innocua, Escherichia coli and Salmonella enteritidis after separation by reversed-phase HPLC. From this fraction a pure peptide was isolated and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). This peptide correspond to the 107–136 fragment of the ␣ chain of bovine hemoglobin. The minimum inhibitory concentrations (MIC) towards the four strains and its hemolytic activity towards bovine erythrocytes were determined. A MIC of 38 M was reported against L. innocua and 76 M for other various bacterial species. This peptide had no hemolytic activity up to 380 M concentration. © 2004 Published by Elsevier Inc. Keywords: Antibacterial peptide; Bovine hemoglobin; Pepsin; Active peptide; Hydrolysate
1. Introduction Mammalian hemoglobins have been described as a source of biologically active peptides with various functions such as opio¨ıd [13], analgesic [19], bacterial growth-stimulating [20], antimicrobial [6,9,14]. These peptides have been generated in vivo in mammalian tissues or in vitro from chemical or enzymatic treated hemoglobins [7,8]. These new active sequences isolated from several hemoglobins are of great interest in the context of research in fundamental biology and biotechnology. A number of active peptides were obtained from peptic bovine hemoglobin hydrolysates. The two first peptides reported were morphine like peptides called hemorphin4 ( 34–37) and hemorphin-5 ( 34–38) [3]. Three other
∗
Corresponding author. Tel.: +33 20 43 4438; fax: +33 20 43 4472. E-mail address: [email protected] (N. Nedjar-Arroume).
0196-9781/$ – see front matter © 2004 Published by Elsevier Inc. doi:10.1016/j.peptides.2004.12.008
opio¨ıd peptides were isolated and characterized in our laboratory: LVV-hemorphin-7 ( 31–40) [15], VV-hemorphin-7 ( 32–40) [15], and VV-hemorphin-4 ( 32–37) [10]. Zhao et al. [21] isolated two other opio¨ıd peptides: LVV-hemorphin-5 ( 31–38) and VV-hemorphin-5 ( 32–38). Peptides with other activities were also obtained from these peptic hydrolysates such as a bradykinin-potentiating peptide [16], two analgesic peptides: the kyotorphin (␣ 140–141) [18] and neokyotorphin (␣ 137–141) [22]. The biologically active peptides derived from bovine hemoglobin could be interesting as components of functional foods. The first identification of antibacterial sequence in bovine hemoglobin was reported in 1999 by Fogac¸a et al. [5] who purified from the gut of the tick Boophilus microplus a peptide corresponding to the 33–61 fragment of the ␣ chain. These authors assumed that proteolytic degradation of hemoglobin resulting in the production of this antibacterial peptide took place inside the tick gut for its own defense against microorganisms.
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R. Daoud et al. / Peptides 26 (2005) 713–719
Recently we have shown that a peptide isolated from a peptic bovine hemoglobin hydrolysate exhibits an antibacterial activity against Micrococcus luteus [6]. This peptide corresponds to a 1–23 sequence. It is the first antibacterial peptide obtained from enzymatic hydrolysis of bovine hemoglobin. Numerous antibacterial peptides are composed of a large amino acid sequence which can adopt a ␣-helical linear structure or a circular structure organized in -sheet. The ␣-helical conformation of active peptides is often essential with regard to their mechanism of action towards the micro-organisms [12]. Such peptides could be forwarded among large appearing at the beginning of proteolysis reaction. In this paper, we report isolation and characterization of a second antibacterial peptide from a peptide hydrolysate obtained by peptic hydrolysis of bovine hemoglobin at low degree of hydrolysis.
2. Materials and methods 2.1. Materials All common chemicals and solvents were of analytical grade from commercial sources. Bovine hemoglobin and pig pepsin were purchased from Sigma Chemicals. Acetonitrile was of HPLC grade. Water was obtained from a Millipore Milli-Q system; the resistance was about 18 M. All aqueous HPLC eluents were bubbled with Waters In Line Degasser. 2.2. Hydrolysate preparations Hemoglobin solution (100 ml) at 1% (w/v) was digested by pig pepsin (EC 3.4.23.1) (40,000 Anson units; Sigma Chemicals) at 23 ◦ C in 0.1 M sodium acetate buffer, pH 4.5, in the presence of urea (5.3 M). The enzymatic hydrolyses were stopped by addition of disodium tetraborate (0.32 M, pH 12.7) up to a final pH of 10. The enzymatic hydrolyses of hemoglobin were performed at different corrected degrees of hydrolysis (DHc) according to the trinitrobenzene sulphonate method [1]. 2.3. HPLC analyzes The liquid chromatographic system consisted of a Waters 600E automated gradient controller pump module, a Waters Wisp 717 automatic sampling device and a Waters 996 photodiode array detector. Spectral and chromatographic data were stored on a NEC image 466 computer. Millennium software was used to plot, acquire and analyze chromatographic data. All the chromatographic processes were performed with a Vydac C4 column (250 mm × 10 mm i.d.). The mobile phase was water/trifluoroacetic acid (1000:1, v/v) as eluent A and acetonitrile/water/trifluoroacetic acid (600:400:1, v/v/v) as eluent B. The flow rate was 5 ml/min. Samples were filtered
through 0.22 m filters and then injected. The gradient applied was 0–67% (v/v) B over 30 min then 67–87% (v/v) B over 35 min. On-line UV absorbance scans were performed between 200 and 300 nm at a rate of one spectrum per second with a resolution of 1.2 nm. Chromatographic analyses were completed with Millennium software. 2.4. Bacteria and antimicrobial assays The antibacterial activity was determined by the method of Parish et al. [14]. Nine bacterial species were used as test micro-organisms for determination of antibacterial activity: three Gramnegative (Escherichia coli, Shiegella sonnei and Salmonella enteritidis) and six Gram-positive (M. luteus A270, Listeria innocua, Enterococcus faecalis, Bacillus cereus, Staphylococcus saprophyticus and Staphylococcus simulans). The nine bacterial species were conserved at −24 ◦ C in glycerol containing nutrient broth and were sub-cultured twice in Muller-Hinton (Biokar Diagnostics) at 30 ◦ C under agitation (60 rpm) for M. luteus A270 and at 37 ◦ C for other bacterial species. The bacterial strains used as indicator bacterial species were isolated from food products in our laboratory, except M. luteus A270. The cells of the these bacterial species were washed twice in 10 mM sodium phosphate buffer pH 6.5 and were then added at a concentration of 107 CFU/plate in 10 mL of the layer containing: Agarose 1%, Triton X100 0.02%, and BSA 0.02%. For M. luteus A270 the layer contained: agar 1%, and Muller-Hinton 21 g/L. The peptide (1 mg/mL) was diluted in 50 mM sodium phosphate buffer, pH 6.5 and was added (20 L) to wells punched in the agarose layer allowed to diffuse in the layer and incubated in a humidified close container during 3 h at 4 ◦ C for M. luteus A270 and at 30 ◦ C for other strains. Following this diffusion period, 10 mL of 1% agar and 42 g/L Muller-Hinton were added as a top layer and plates were incubated 24 h at 37 ◦ C. With M. luteus A270, no top layer was used and plates were incubated 24 h at 30 ◦ C. The antibacterial activity was measured as the diameter of clear zone of growth inhibition by comparison to a positive control which was Chloramphenicol, and to a negative control: sodium phosphate buffer. The minimum inhibitory concentration (MIC) of the peptide was determined in a microtiter plate assay system. Each well of the microtiter plate contained 50 L of onefold concentrated Mueller-Hinton broth inoculated with ca. 106 CFU mL−1 and 50 L of the peptide at serial two-fold dilutions in phosphate buffer 50 mM pH 5.5 (from 1 to 0.0625 mg/mL). As a turbidity control, bacterial strains were incubated as described above without peptide. The MIC was determined as the lowest concentration of the peptide that completely inhibited growth of the indicator bacterial species after 24 h of incubation at 30 or 37 ◦ C. Growth was measured spectrophotometrically at 540 nm in a MRX Thermolab Systems microplate reader.
R. Daoud et al. / Peptides 26 (2005) 713–719
The mode of action of the peptide was also determined. Hundred microliters of cell suspension were distributed in a microtiter plate well and 100 L of the peptide were added. Cells were enumerated on nutrient agar medium before and after 24 h of incubation at 30 or 37 ◦ C. 2.5. Amino acid analyzes Amino acids were analyzed with a Waters Picotag Workstation. Peptide hydrolysis was achieved with constant boiling (6 M) HCl containing 1% (w/v) phenol, for 24 h at 110 ◦ C. The precolumn derivatization of amino acids with phenyl isothiocyannate and the HPLC separation of derivatized amino acids on a waters reverse-phase Picotag column (150 mm × 3.9 mm i.d.) were performed by the method of Bidlingmeyer et al. [2]. The detection wavelength was 254 nm and the flow rate was 1 mL/min. 2.6. Mass spectrometry analysis MS and MS/MS measurements of the active peptide were performed in positive ion mode using MALDI and ESIMS/MS, respectively [6]. MALDI/MS experiments were carried out on a Finnigan Vision 2000 reflectron time-of-flight instrument (Finnigan MAT, Bremen, Germany), equipped with a microscope and a video camera. MS and MS/MS measurements of the active peptide were performed in positive ion mode using electrospray ionization (ESI) and ESI-MS/MS, respectively. ESI mass spectrometry was performed using a triple quadrupole instrument Applied Biosystems API 3000 (PE Sciex, Toronto, Canada) equipped with an electrospray ion source. 2.7. Hemolytic activity The hemolytic activity of the peptide was determined by methods of Strub et al. [17] and of Dathe et al. [4], slightly modified. 5 mL of bovine blood were centrifuged at 3500 rpm during 10 min to isolate erythrocytes which were washed three times with 10 mM sodium phosphate pH 7.5 containing NaCl 9 g/L (NaCl/Pi). The cell concentration stock suspension was adjusted to 109 cells/mL. The cell suspension (12 L) varying amounts of peptide stock solution and the buffer were pipetted into eppendorf tubes to give a final volume of 50 L. Then, the eppendorf tubes with 2.5.108 cells/mL were incubated at 37 ◦ C during 40 min. After centrifugation (5000 rpm, 5 min), 30 L of supernatant was diluted in 500 L water. The absorbance of the diluted solution was measured at 420 nm. The absorbance obtained after treating erythrocytes with only NaCl/Pi and SDS (0.2%) was taken as 0% and 100%, respectively. A large of experiments was carried out to check their reproducibility.
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3. Results and discussion 3.1. Peptic hydrolysis of hemoglobin Kinetics studies of peptic hydrolysis of hemoglobin were followed at different degrees of hydrolysis (3%, 5.5%, 8%, 16.5% and 18.5%) in order to generate a maximum of intermediate peptides of medium size. These hydrolyses were carried out at 23 ◦ C in 0.1 M sodium acetate buffer at pH 4.5 with urea denatured hemoglobin. The resulting hydrolysates were analyzed in one step by reversed phase HPLC on a C4column according to a method described previously [10]. Fig. 1 shows the chromatograms of peptic hydrolysates of hemoglobin obtained under these conditions according to the degree of hydrolysis. All the denatured hemoglobin molecules were rapidly converted to intermediate peptides, which were then degraded more slowly to final peptides. This type of kinetic already observed in the course of hydrolysis of denatured protein corresponds to a “zipper” mechanism [11]. A peptide group was observed at low degree of hydrolysis (around 3%) and disappeared rapidly. This group was called fraction A in Fig. 1. Its retention time lays from 35 to 38 min. This fraction which contains large intermediate peptides was used for researching antibacterial activities. 3.2. Isolation of an antimicrobial peptide The peptic hydrolysis of denatured hemoglobin was performed for 2.5 min which corresponds to 3% of degree of hydrolysis. The fraction A was prepared in one step by semipreparative reversed phase HPLC. The antibacterial activity of this fraction A was tested in Petri plates against nine bacterial species: three Gramnegative (E. coli, S. sonnei and S. enteritidis) and six Grampositive (M. luteus A270, L. innocua, E. faecalis, B. cereus, S. saprophyticus and S. simulans). The antibacterial activity was measured as the diameter of clear zone of growth inhibition. At the tested concentrations (1 mg/mL), the fraction A exhibited an antibacterial activity towards M. luteus A270, L. innocua, E. coli and S. enteritidis (Table 1). Table 1 Antibacterial activity of fraction A obtained from the peptic hydrolysis of denatured hemoglobin at 3% of degree of hydrolysis Bacterial species
Growth inhibition of fraction A
M. luteus (Gram-positive) L. innocua (Gram-positive) B. cereus (Gram-positive) E. faecalis (Gram-positive) S. simulans (Gram-positive) S. saprophyticus (Gram-positive) E. coli (Gram-negative) S. sonnei (Gram-negative) S. enteritidis (Gram-negative)
+ + − − − − + − +
Fraction concentration was 1 mg/mL.
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Fig. 1. HPLC chromatograms of the denatured hemoglobin hydrolysate. Kinetics studies of peptic hydrolysis of hemoglobin were followed at different of corrected degrees of hydrolysis (DHc) and at 23 ◦ C in 0.1 M sodium acetate buffer at pH 4.5. The hydrolysates were analyzed by RP-HPLC on a C4 column with the conditions described in Section 2.
These results show that the fraction A is active. As it contains many peptides, a second reverse phase-HPLC of this fraction was performed in order to try to obtain pure peptides (Fig. 2). The chromatograms shows a high peak corresponding to a pure peptide as indicated by millennium software. This peptide had a retention time of 37 min. Its antibacterial activity was tested in Petri plate against these four strains. The diameters of inhibition of this peptide towards M. luteus
A270, L. innocua, E. coli and S. enteridis were, respectively, of 15, 25, 17 and 16 mm (Table 2). 3.3. Characterization of the active peptide This peptide was identified by amino acid analysis and MALDI/MS studies. Fig. 3 shows the positive Maldi spectrum of this peptide. The accurate relative molecular mass of
R. Daoud et al. / Peptides 26 (2005) 713–719
717
Fig. 2. HPLC chromatogram of the fraction A. The fraction A was analyzed by RP-HPLC on a C4 column with the conditions described in Section 2. The pure peptide with a retention time of 37 min is marked by arrow. Table 2 Determination of the diameter of the inhibition zone and the minimum inhibitory concentration (MIC) Bacterial species
Diameters inhibition (mm)
MIC (M)
M. luteus (Gram-positive) L. innocua (Gram-positive) E. coli (Gram-negative) S. enteritidis (Gram-negative)
15 25 17 16
76 38 76 76
the peptide, deduced from the m/z value of (M + H)+ by substraction of one mass unit for the attached proton is 3150 Da. According to this molecular mass and taking into account its amino acid composition obtained by Picotag amino acid analysis (Table 3) and the amino acid sequence of ␣ and  bovine hemoglobin chains, it was deduced that this peptide corresponds either to the 107–136 fragment or the 106–135
fragment of the ␣ chain which have the same amino acid composition. In order to resolve this ambiguity, tandem MS was performed. In the collision cell of the mass spectrometer, the precursor ion fragments by CID into structurally significant product ions. Fig. 4 shows the CID spectrum of the (M + H)+ ion and displays notation of fragment ions of the peptide according to this CID spectrum. Interpretation along the fragmentation process results in the recognition of a series of ions indicating the following sequence: 107 Val-Thr-Leu-Ala-Ser-His-Leu-Pro-Ser-Asp-Phe-ThrPro-Ala-Val-His-Ala-Ser-Leu-Asp-Lys-Phe-Leu-Ala-AsnVal-Ser-Thr-Val-Leu136 . Owing to this amino acid sequence, this peptide effectively represents the 107–136 fragment of the ␣ chain of bovine hemoglobin.
Fig. 3. MALDI/MS of fraction with retention time of 37 min isolated by RP-HPLC. The ion at m/z 3151 designated as a molecular cation (M + H)+ , suggests residues 107–136 of the hemoglobin ␣ chain.
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Fig. 4. CID spectrum of (M + H)+ ion of ␣ 107–136 peptide isolated from peptic hydrolysate of bovine hemoglobin, and notation of fragment ions.
3.4. Determination of the minimum inhibitory concentrations of ␣ 107–136 peptide MIC of the ␣ 107–136 peptide was determined in a microtiter plate assay system towards M. luteus A270, L. innocua, E. coli and S. enteritidis (Table 2). It was the lowest concentration of the peptide that completely inhibited growth of the strain after an incubation of 24 h. The initial population was 106 CFU/mL. After 24 h incubation with varying concentrations of peptide, we determined the MIC. Table 2 shows the MIC of the ␣ 107–136 peptide obtained for these bacterial species. The MIC were 76 M towards M. luteus A270, E. coli and S. enteritidis and 38 M towards L. innocua. This last MIC is twice as low as MIC observed with other bacterial species. The MIC of this peptide towards M. luteus A270 is about height times as low as the MIC of the peptide ␣ 1–23 which was previously isolated from a peptic hydrolysate of bovine hemoglobin (6). An average value of 30% was deduced Table 3 Amino acid composition of ␣ 107–136 peptide isolated from peptic hydrolysate of bovine hemoglobin Amino acid
Peptide
Asp Glu Ser Gly His Arg Thr Ala Pro Tyr Val Met Cys Ile Leu Phe Lys Trp
2.96 0 4.05 0 2.00 0 2.99 4.05 1.96 0 3.98 0 0 0 5.03 1.98 0.97 ND
Amino acids are expressed in residues per molecules. ND indicates that individual amino acids were not determined.
for the ␣-helical content of this peptide by different mathematical models of the Network Protein Sequence analysis Internet server of the Pole Bio-informatique Lyonnais (http://pbil.ibcp.fr). From the same method an average value of 54% of ␣-helical content was deduced for the ␣ 107–136 peptide. This higher ␣-helical content could to explain the higher antibacterial activity of this last peptide compared to ␣ 1–23 peptide. At a concentration corresponding to the MIC, cells enumeration indicated a bactericidal effect of the ␣ 107–136 peptide: after 24 h of incubation, cell population was 2.86104 CFU/mL whereas initial cell population was 106 CFU/mL. 3.5. Hemolytic activity of ␣ 107–136 peptide The hemolytic activity of this peptide was tested towards bovine erythrocytes. Several peptide concentrations from 76 to 380 M were tested. No hemolytic was observed with peptide concentrations up to 380 M, this last concentration corresponds to 5 or 10 times the MIC (76 or 38 M). With the ␣ 1–23 peptide, no hemolysis of erythrocyte was observed with peptide concentration up to three times the MIC and 54% hemolysis was observed with peptide concentration corresponding to 5 times the MIC. These results show that the ␣ 107–136 peptide would be used at concentrations up to 5 or 10 times the MIC (380 M), a very high concentration for a peptide.
4. Conclusion The ␣ 107–136 peptide is the second antimicrobial peptide obtained by in vitro proteolysis of bovine hemoglobin. This peptide is active against four bacterial species: M. luteus A270, L. innocua, E. coli and S. enteritidis. Whatever the bacterial species, at a concentration corresponding to its MIC, cells enumeration indicated a bactericidal effect of this peptide. Moreover, ␣ 107–136 peptide would be used at concentrations up to 5 or 10 times the MIC depending on the bacterial species.
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In the current context of food safety and food protection by means of natural products, such an antibacterial peptide derived from bovine hemoglobin would be interesting as a preservative for storage and distribution of in meat based products. Research is in progress to separate the intermediate peptides of the fraction A testing their antibacterial activities.
[10]
[11]
Acknowledgement
[12]
The authors thank Christine Vanuxem from University of Lille I, for assistance in the preparation of this manuscript.
[13]
References [1] Adler-Nissen J. Determination of the degree of hydrolysis of food protein hydrolysats by tribenzenesulfonic acid. J Agric Food Chem 1979;27:1256–62. [2] Bidlingmeyer BA, Cohen SA, Tarvin TL, Napier JR, Hancock WS. Rapid analysis of amino acids using pre-column derivatization. J Chromatogr 1984;336:93–104. [3] Brantl V, Gramsch C, Lottspeich F, Mertz R, Jaeger KH, Herz A. Novel opioid peptides derived from hemoglobin: hemorphins. Eur J Pharmacol 1986;125:309–10. [4] Dathe M, Schumann M, Wieprecht T, Winkler A, Beyermann M, Krause E, et al. Peptide helicity and membrane surface charge modulate the balance of electrostatic and hydrophobic interactions with lipid bilayers and biological membranes. Biochemistry 1996;35:12612–22. [5] Fogac¸a AC, Da Silva PI, Teresa Jr M, Miranda M, Bianchi AG, Miranda A, et al. Antimicrobial activity of a bovine hemoglobin fragment in the tick Boophilus microplus. J Biol Chem 1999;274:25330–4. [6] Froidevaux R, Krier F, Nedjar-Arroume N, Vercaigne-Marko D, Kosciarz E, Ruckebusch C, et al. Antibacterial activity of a pepsinderived bovine hemoglobin fragment. FEBS Lett 2001;491:159–63. [7] Ivanov VT, Karelin AA, Philippova MM, Nazimov IV, Pletnev VZ. Hemoglobin as a source of endogenous bioactive peptides: the concept of tissue-specific peptide pool. Biopoly 1997;43:171–88. [8] Karelin AA, Philippova MM, Ivanov VT. Proteolytic degradation of haemoglobin in erythrocytes leads to biologically active peptides. Peptides 1995;16:693–7. [9] Liepke C, Baxmann S, Heine C, Breithaupt N, St¨andker L, Forssmann WG. Human hemoglobin-derived peptides exhibit antimicro-
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
719
bial activity: a class of host defense peptides. J Chromatogr B 2003;791:345–56. Lignot B, Froidevaux R, Nedjar-Arroume N, Guillochon D. Solvent effect on kinetics of appearance of neokyotorphin VVh4 and a bradykinin-potentiating peptide in the course of peptic hydrolysis of bovine haemoglobin. Biotechnol Appl Biochem 1999;30:201– 7. Lindertrom-lang K. Proteins and enzymes. III. Lane medical lectures, vol. VI. Stanford, CA: Stanford University Press; 1952. p. 53– 72. Nicolas P, Mor A. Peptides as weapons against microorganisms in the chemical defense system of vertebrates. Annu Rev Microbiol 1995;49:277–304. Nyberg F, Sanderson K, Gl¨amsta EL. The hemorphins: a new class of opioid peptides derived from the blood protein haemoglobin. Biopoly 1997;43:147–56. Parish CA, Jiang H, Tokiwa Y, Berova N, Nakanishi K, Mc Cabe D, et al. Broad-spectrum antimicrobial activity of hemoglobin. Bioorg Med Chem 2001;9:377–82. Piot JM, Zhao QY, Guillochon D, Ricart G, Thomas D. Isolation and characterisation of two opioid peptides from a bovine hemoglobin peptic hydrolysate. Biochem Biophys Res Commun 1992;189:101–10. Piot JM, Zhao QY, Guillochon D, Ricart G, Thomas D. Isolation and characterisation of a bradykinin-potentiating peptide from bovine peptic hemoglobine hydrolysate. FEBS Lett 1992;299:75–9. Strub JM, Goumon Y, Lugardon K, Capon C, Lopez M, Moniatte M, et al. Antibacterial activity of glycosylated and phosphorylated chromogranin A-derived peptide 173–194 from bovine adrenal medullary chromaffin granules. J Biol Chem 1996;271:28533–40. Takagi H, Shiomi H, Ueda H, Amano H. A novel analgesic dipeptide from bovine brain is a possible Met-enkephalin realeaser. Nature 1979;5737:410–2. Takagi H, Shiomi H, Hayashi K, Kiso Y, Kitagawa K. Isolation of a novel analgesic pentapeptide, neo-kyotorphin, from bovine brain. Life Sci 1982;31:1733–6. Zhao QY, Piot JM, Gautier V, Cottenceau G. Peptic peptide mapping by HPLC, on line with photodiode array detection, of a haemoglobin hydrolysate produced at pilot-plant scale from an ultrafiltration process. Appl Microbiol Biotechnol 1996;45:778–84. Zhao QY, Sannier F, Piot JM. Kinetics of appearance of four hemorphins from bovine hemoglobin peptic hydrolysates by HPLC coupled with photodiode array detection. Biochem Biophys Acta 1996;1295:73–80. Zhao QY, Molina P, Piot JM. Peptic peptide mapping by HPLC, on line with photodiode array detection, of a haemoglobin hydrolysate produced at pilot-plant scale from an ultrafiltration process. J Liq Chrom Rel Technol 1997;20:1717–39.
New antibacterial peptide derived from bovine hemoglobin Rachid Daoud a , Veronique Dubois a , Loredana Bors-Dodita a , Naima Nedjar-Arroume a,∗ , Francois Krier a , Nour-Eddine Chihib a , Patrice Mary a , Mostafa Kouach b , Gilbert Briand b , Didier Guillochon a b
a Laboratoire de Technologie des Substances Naturelles, IUT “A” Lille I, BP 179, 59653 Villeneuve d’Ascq Cedex, France Laboratoire d’Application de Spectrom´etrie de Masse, Service Commun de Physicochimie, Facult´e de M´edecine H. Warembourg, Pˆole Recherche, Place de Verdun, Lille II, France
Received 20 October 2004; received in revised form 10 December 2004; accepted 14 December 2004 Available online 19 March 2005
Abstract Peptic digestion of bovine hemoglobin at low degree of hydrolysis yields an intermediate peptide fraction exhibiting antibacterial activity against Micrococcus luteus A270, Listeria innocua, Escherichia coli and Salmonella enteritidis after separation by reversed-phase HPLC. From this fraction a pure peptide was isolated and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). This peptide correspond to the 107–136 fragment of the ␣ chain of bovine hemoglobin. The minimum inhibitory concentrations (MIC) towards the four strains and its hemolytic activity towards bovine erythrocytes were determined. A MIC of 38 M was reported against L. innocua and 76 M for other various bacterial species. This peptide had no hemolytic activity up to 380 M concentration. © 2004 Published by Elsevier Inc. Keywords: Antibacterial peptide; Bovine hemoglobin; Pepsin; Active peptide; Hydrolysate
1. Introduction Mammalian hemoglobins have been described as a source of biologically active peptides with various functions such as opio¨ıd [13], analgesic [19], bacterial growth-stimulating [20], antimicrobial [6,9,14]. These peptides have been generated in vivo in mammalian tissues or in vitro from chemical or enzymatic treated hemoglobins [7,8]. These new active sequences isolated from several hemoglobins are of great interest in the context of research in fundamental biology and biotechnology. A number of active peptides were obtained from peptic bovine hemoglobin hydrolysates. The two first peptides reported were morphine like peptides called hemorphin4 ( 34–37) and hemorphin-5 ( 34–38) [3]. Three other
∗
Corresponding author. Tel.: +33 20 43 4438; fax: +33 20 43 4472. E-mail address: [email protected] (N. Nedjar-Arroume).
0196-9781/$ – see front matter © 2004 Published by Elsevier Inc. doi:10.1016/j.peptides.2004.12.008
opio¨ıd peptides were isolated and characterized in our laboratory: LVV-hemorphin-7 ( 31–40) [15], VV-hemorphin-7 ( 32–40) [15], and VV-hemorphin-4 ( 32–37) [10]. Zhao et al. [21] isolated two other opio¨ıd peptides: LVV-hemorphin-5 ( 31–38) and VV-hemorphin-5 ( 32–38). Peptides with other activities were also obtained from these peptic hydrolysates such as a bradykinin-potentiating peptide [16], two analgesic peptides: the kyotorphin (␣ 140–141) [18] and neokyotorphin (␣ 137–141) [22]. The biologically active peptides derived from bovine hemoglobin could be interesting as components of functional foods. The first identification of antibacterial sequence in bovine hemoglobin was reported in 1999 by Fogac¸a et al. [5] who purified from the gut of the tick Boophilus microplus a peptide corresponding to the 33–61 fragment of the ␣ chain. These authors assumed that proteolytic degradation of hemoglobin resulting in the production of this antibacterial peptide took place inside the tick gut for its own defense against microorganisms.
714
R. Daoud et al. / Peptides 26 (2005) 713–719
Recently we have shown that a peptide isolated from a peptic bovine hemoglobin hydrolysate exhibits an antibacterial activity against Micrococcus luteus [6]. This peptide corresponds to a 1–23 sequence. It is the first antibacterial peptide obtained from enzymatic hydrolysis of bovine hemoglobin. Numerous antibacterial peptides are composed of a large amino acid sequence which can adopt a ␣-helical linear structure or a circular structure organized in -sheet. The ␣-helical conformation of active peptides is often essential with regard to their mechanism of action towards the micro-organisms [12]. Such peptides could be forwarded among large appearing at the beginning of proteolysis reaction. In this paper, we report isolation and characterization of a second antibacterial peptide from a peptide hydrolysate obtained by peptic hydrolysis of bovine hemoglobin at low degree of hydrolysis.
2. Materials and methods 2.1. Materials All common chemicals and solvents were of analytical grade from commercial sources. Bovine hemoglobin and pig pepsin were purchased from Sigma Chemicals. Acetonitrile was of HPLC grade. Water was obtained from a Millipore Milli-Q system; the resistance was about 18 M. All aqueous HPLC eluents were bubbled with Waters In Line Degasser. 2.2. Hydrolysate preparations Hemoglobin solution (100 ml) at 1% (w/v) was digested by pig pepsin (EC 3.4.23.1) (40,000 Anson units; Sigma Chemicals) at 23 ◦ C in 0.1 M sodium acetate buffer, pH 4.5, in the presence of urea (5.3 M). The enzymatic hydrolyses were stopped by addition of disodium tetraborate (0.32 M, pH 12.7) up to a final pH of 10. The enzymatic hydrolyses of hemoglobin were performed at different corrected degrees of hydrolysis (DHc) according to the trinitrobenzene sulphonate method [1]. 2.3. HPLC analyzes The liquid chromatographic system consisted of a Waters 600E automated gradient controller pump module, a Waters Wisp 717 automatic sampling device and a Waters 996 photodiode array detector. Spectral and chromatographic data were stored on a NEC image 466 computer. Millennium software was used to plot, acquire and analyze chromatographic data. All the chromatographic processes were performed with a Vydac C4 column (250 mm × 10 mm i.d.). The mobile phase was water/trifluoroacetic acid (1000:1, v/v) as eluent A and acetonitrile/water/trifluoroacetic acid (600:400:1, v/v/v) as eluent B. The flow rate was 5 ml/min. Samples were filtered
through 0.22 m filters and then injected. The gradient applied was 0–67% (v/v) B over 30 min then 67–87% (v/v) B over 35 min. On-line UV absorbance scans were performed between 200 and 300 nm at a rate of one spectrum per second with a resolution of 1.2 nm. Chromatographic analyses were completed with Millennium software. 2.4. Bacteria and antimicrobial assays The antibacterial activity was determined by the method of Parish et al. [14]. Nine bacterial species were used as test micro-organisms for determination of antibacterial activity: three Gramnegative (Escherichia coli, Shiegella sonnei and Salmonella enteritidis) and six Gram-positive (M. luteus A270, Listeria innocua, Enterococcus faecalis, Bacillus cereus, Staphylococcus saprophyticus and Staphylococcus simulans). The nine bacterial species were conserved at −24 ◦ C in glycerol containing nutrient broth and were sub-cultured twice in Muller-Hinton (Biokar Diagnostics) at 30 ◦ C under agitation (60 rpm) for M. luteus A270 and at 37 ◦ C for other bacterial species. The bacterial strains used as indicator bacterial species were isolated from food products in our laboratory, except M. luteus A270. The cells of the these bacterial species were washed twice in 10 mM sodium phosphate buffer pH 6.5 and were then added at a concentration of 107 CFU/plate in 10 mL of the layer containing: Agarose 1%, Triton X100 0.02%, and BSA 0.02%. For M. luteus A270 the layer contained: agar 1%, and Muller-Hinton 21 g/L. The peptide (1 mg/mL) was diluted in 50 mM sodium phosphate buffer, pH 6.5 and was added (20 L) to wells punched in the agarose layer allowed to diffuse in the layer and incubated in a humidified close container during 3 h at 4 ◦ C for M. luteus A270 and at 30 ◦ C for other strains. Following this diffusion period, 10 mL of 1% agar and 42 g/L Muller-Hinton were added as a top layer and plates were incubated 24 h at 37 ◦ C. With M. luteus A270, no top layer was used and plates were incubated 24 h at 30 ◦ C. The antibacterial activity was measured as the diameter of clear zone of growth inhibition by comparison to a positive control which was Chloramphenicol, and to a negative control: sodium phosphate buffer. The minimum inhibitory concentration (MIC) of the peptide was determined in a microtiter plate assay system. Each well of the microtiter plate contained 50 L of onefold concentrated Mueller-Hinton broth inoculated with ca. 106 CFU mL−1 and 50 L of the peptide at serial two-fold dilutions in phosphate buffer 50 mM pH 5.5 (from 1 to 0.0625 mg/mL). As a turbidity control, bacterial strains were incubated as described above without peptide. The MIC was determined as the lowest concentration of the peptide that completely inhibited growth of the indicator bacterial species after 24 h of incubation at 30 or 37 ◦ C. Growth was measured spectrophotometrically at 540 nm in a MRX Thermolab Systems microplate reader.
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The mode of action of the peptide was also determined. Hundred microliters of cell suspension were distributed in a microtiter plate well and 100 L of the peptide were added. Cells were enumerated on nutrient agar medium before and after 24 h of incubation at 30 or 37 ◦ C. 2.5. Amino acid analyzes Amino acids were analyzed with a Waters Picotag Workstation. Peptide hydrolysis was achieved with constant boiling (6 M) HCl containing 1% (w/v) phenol, for 24 h at 110 ◦ C. The precolumn derivatization of amino acids with phenyl isothiocyannate and the HPLC separation of derivatized amino acids on a waters reverse-phase Picotag column (150 mm × 3.9 mm i.d.) were performed by the method of Bidlingmeyer et al. [2]. The detection wavelength was 254 nm and the flow rate was 1 mL/min. 2.6. Mass spectrometry analysis MS and MS/MS measurements of the active peptide were performed in positive ion mode using MALDI and ESIMS/MS, respectively [6]. MALDI/MS experiments were carried out on a Finnigan Vision 2000 reflectron time-of-flight instrument (Finnigan MAT, Bremen, Germany), equipped with a microscope and a video camera. MS and MS/MS measurements of the active peptide were performed in positive ion mode using electrospray ionization (ESI) and ESI-MS/MS, respectively. ESI mass spectrometry was performed using a triple quadrupole instrument Applied Biosystems API 3000 (PE Sciex, Toronto, Canada) equipped with an electrospray ion source. 2.7. Hemolytic activity The hemolytic activity of the peptide was determined by methods of Strub et al. [17] and of Dathe et al. [4], slightly modified. 5 mL of bovine blood were centrifuged at 3500 rpm during 10 min to isolate erythrocytes which were washed three times with 10 mM sodium phosphate pH 7.5 containing NaCl 9 g/L (NaCl/Pi). The cell concentration stock suspension was adjusted to 109 cells/mL. The cell suspension (12 L) varying amounts of peptide stock solution and the buffer were pipetted into eppendorf tubes to give a final volume of 50 L. Then, the eppendorf tubes with 2.5.108 cells/mL were incubated at 37 ◦ C during 40 min. After centrifugation (5000 rpm, 5 min), 30 L of supernatant was diluted in 500 L water. The absorbance of the diluted solution was measured at 420 nm. The absorbance obtained after treating erythrocytes with only NaCl/Pi and SDS (0.2%) was taken as 0% and 100%, respectively. A large of experiments was carried out to check their reproducibility.
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3. Results and discussion 3.1. Peptic hydrolysis of hemoglobin Kinetics studies of peptic hydrolysis of hemoglobin were followed at different degrees of hydrolysis (3%, 5.5%, 8%, 16.5% and 18.5%) in order to generate a maximum of intermediate peptides of medium size. These hydrolyses were carried out at 23 ◦ C in 0.1 M sodium acetate buffer at pH 4.5 with urea denatured hemoglobin. The resulting hydrolysates were analyzed in one step by reversed phase HPLC on a C4column according to a method described previously [10]. Fig. 1 shows the chromatograms of peptic hydrolysates of hemoglobin obtained under these conditions according to the degree of hydrolysis. All the denatured hemoglobin molecules were rapidly converted to intermediate peptides, which were then degraded more slowly to final peptides. This type of kinetic already observed in the course of hydrolysis of denatured protein corresponds to a “zipper” mechanism [11]. A peptide group was observed at low degree of hydrolysis (around 3%) and disappeared rapidly. This group was called fraction A in Fig. 1. Its retention time lays from 35 to 38 min. This fraction which contains large intermediate peptides was used for researching antibacterial activities. 3.2. Isolation of an antimicrobial peptide The peptic hydrolysis of denatured hemoglobin was performed for 2.5 min which corresponds to 3% of degree of hydrolysis. The fraction A was prepared in one step by semipreparative reversed phase HPLC. The antibacterial activity of this fraction A was tested in Petri plates against nine bacterial species: three Gramnegative (E. coli, S. sonnei and S. enteritidis) and six Grampositive (M. luteus A270, L. innocua, E. faecalis, B. cereus, S. saprophyticus and S. simulans). The antibacterial activity was measured as the diameter of clear zone of growth inhibition. At the tested concentrations (1 mg/mL), the fraction A exhibited an antibacterial activity towards M. luteus A270, L. innocua, E. coli and S. enteritidis (Table 1). Table 1 Antibacterial activity of fraction A obtained from the peptic hydrolysis of denatured hemoglobin at 3% of degree of hydrolysis Bacterial species
Growth inhibition of fraction A
M. luteus (Gram-positive) L. innocua (Gram-positive) B. cereus (Gram-positive) E. faecalis (Gram-positive) S. simulans (Gram-positive) S. saprophyticus (Gram-positive) E. coli (Gram-negative) S. sonnei (Gram-negative) S. enteritidis (Gram-negative)
+ + − − − − + − +
Fraction concentration was 1 mg/mL.
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Fig. 1. HPLC chromatograms of the denatured hemoglobin hydrolysate. Kinetics studies of peptic hydrolysis of hemoglobin were followed at different of corrected degrees of hydrolysis (DHc) and at 23 ◦ C in 0.1 M sodium acetate buffer at pH 4.5. The hydrolysates were analyzed by RP-HPLC on a C4 column with the conditions described in Section 2.
These results show that the fraction A is active. As it contains many peptides, a second reverse phase-HPLC of this fraction was performed in order to try to obtain pure peptides (Fig. 2). The chromatograms shows a high peak corresponding to a pure peptide as indicated by millennium software. This peptide had a retention time of 37 min. Its antibacterial activity was tested in Petri plate against these four strains. The diameters of inhibition of this peptide towards M. luteus
A270, L. innocua, E. coli and S. enteridis were, respectively, of 15, 25, 17 and 16 mm (Table 2). 3.3. Characterization of the active peptide This peptide was identified by amino acid analysis and MALDI/MS studies. Fig. 3 shows the positive Maldi spectrum of this peptide. The accurate relative molecular mass of
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Fig. 2. HPLC chromatogram of the fraction A. The fraction A was analyzed by RP-HPLC on a C4 column with the conditions described in Section 2. The pure peptide with a retention time of 37 min is marked by arrow. Table 2 Determination of the diameter of the inhibition zone and the minimum inhibitory concentration (MIC) Bacterial species
Diameters inhibition (mm)
MIC (M)
M. luteus (Gram-positive) L. innocua (Gram-positive) E. coli (Gram-negative) S. enteritidis (Gram-negative)
15 25 17 16
76 38 76 76
the peptide, deduced from the m/z value of (M + H)+ by substraction of one mass unit for the attached proton is 3150 Da. According to this molecular mass and taking into account its amino acid composition obtained by Picotag amino acid analysis (Table 3) and the amino acid sequence of ␣ and  bovine hemoglobin chains, it was deduced that this peptide corresponds either to the 107–136 fragment or the 106–135
fragment of the ␣ chain which have the same amino acid composition. In order to resolve this ambiguity, tandem MS was performed. In the collision cell of the mass spectrometer, the precursor ion fragments by CID into structurally significant product ions. Fig. 4 shows the CID spectrum of the (M + H)+ ion and displays notation of fragment ions of the peptide according to this CID spectrum. Interpretation along the fragmentation process results in the recognition of a series of ions indicating the following sequence: 107 Val-Thr-Leu-Ala-Ser-His-Leu-Pro-Ser-Asp-Phe-ThrPro-Ala-Val-His-Ala-Ser-Leu-Asp-Lys-Phe-Leu-Ala-AsnVal-Ser-Thr-Val-Leu136 . Owing to this amino acid sequence, this peptide effectively represents the 107–136 fragment of the ␣ chain of bovine hemoglobin.
Fig. 3. MALDI/MS of fraction with retention time of 37 min isolated by RP-HPLC. The ion at m/z 3151 designated as a molecular cation (M + H)+ , suggests residues 107–136 of the hemoglobin ␣ chain.
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Fig. 4. CID spectrum of (M + H)+ ion of ␣ 107–136 peptide isolated from peptic hydrolysate of bovine hemoglobin, and notation of fragment ions.
3.4. Determination of the minimum inhibitory concentrations of ␣ 107–136 peptide MIC of the ␣ 107–136 peptide was determined in a microtiter plate assay system towards M. luteus A270, L. innocua, E. coli and S. enteritidis (Table 2). It was the lowest concentration of the peptide that completely inhibited growth of the strain after an incubation of 24 h. The initial population was 106 CFU/mL. After 24 h incubation with varying concentrations of peptide, we determined the MIC. Table 2 shows the MIC of the ␣ 107–136 peptide obtained for these bacterial species. The MIC were 76 M towards M. luteus A270, E. coli and S. enteritidis and 38 M towards L. innocua. This last MIC is twice as low as MIC observed with other bacterial species. The MIC of this peptide towards M. luteus A270 is about height times as low as the MIC of the peptide ␣ 1–23 which was previously isolated from a peptic hydrolysate of bovine hemoglobin (6). An average value of 30% was deduced Table 3 Amino acid composition of ␣ 107–136 peptide isolated from peptic hydrolysate of bovine hemoglobin Amino acid
Peptide
Asp Glu Ser Gly His Arg Thr Ala Pro Tyr Val Met Cys Ile Leu Phe Lys Trp
2.96 0 4.05 0 2.00 0 2.99 4.05 1.96 0 3.98 0 0 0 5.03 1.98 0.97 ND
Amino acids are expressed in residues per molecules. ND indicates that individual amino acids were not determined.
for the ␣-helical content of this peptide by different mathematical models of the Network Protein Sequence analysis Internet server of the Pole Bio-informatique Lyonnais (http://pbil.ibcp.fr). From the same method an average value of 54% of ␣-helical content was deduced for the ␣ 107–136 peptide. This higher ␣-helical content could to explain the higher antibacterial activity of this last peptide compared to ␣ 1–23 peptide. At a concentration corresponding to the MIC, cells enumeration indicated a bactericidal effect of the ␣ 107–136 peptide: after 24 h of incubation, cell population was 2.86104 CFU/mL whereas initial cell population was 106 CFU/mL. 3.5. Hemolytic activity of ␣ 107–136 peptide The hemolytic activity of this peptide was tested towards bovine erythrocytes. Several peptide concentrations from 76 to 380 M were tested. No hemolytic was observed with peptide concentrations up to 380 M, this last concentration corresponds to 5 or 10 times the MIC (76 or 38 M). With the ␣ 1–23 peptide, no hemolysis of erythrocyte was observed with peptide concentration up to three times the MIC and 54% hemolysis was observed with peptide concentration corresponding to 5 times the MIC. These results show that the ␣ 107–136 peptide would be used at concentrations up to 5 or 10 times the MIC (380 M), a very high concentration for a peptide.
4. Conclusion The ␣ 107–136 peptide is the second antimicrobial peptide obtained by in vitro proteolysis of bovine hemoglobin. This peptide is active against four bacterial species: M. luteus A270, L. innocua, E. coli and S. enteritidis. Whatever the bacterial species, at a concentration corresponding to its MIC, cells enumeration indicated a bactericidal effect of this peptide. Moreover, ␣ 107–136 peptide would be used at concentrations up to 5 or 10 times the MIC depending on the bacterial species.
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In the current context of food safety and food protection by means of natural products, such an antibacterial peptide derived from bovine hemoglobin would be interesting as a preservative for storage and distribution of in meat based products. Research is in progress to separate the intermediate peptides of the fraction A testing their antibacterial activities.
[10]
[11]
Acknowledgement
[12]
The authors thank Christine Vanuxem from University of Lille I, for assistance in the preparation of this manuscript.
[13]
References [1] Adler-Nissen J. Determination of the degree of hydrolysis of food protein hydrolysats by tribenzenesulfonic acid. J Agric Food Chem 1979;27:1256–62. [2] Bidlingmeyer BA, Cohen SA, Tarvin TL, Napier JR, Hancock WS. Rapid analysis of amino acids using pre-column derivatization. J Chromatogr 1984;336:93–104. [3] Brantl V, Gramsch C, Lottspeich F, Mertz R, Jaeger KH, Herz A. Novel opioid peptides derived from hemoglobin: hemorphins. Eur J Pharmacol 1986;125:309–10. [4] Dathe M, Schumann M, Wieprecht T, Winkler A, Beyermann M, Krause E, et al. Peptide helicity and membrane surface charge modulate the balance of electrostatic and hydrophobic interactions with lipid bilayers and biological membranes. Biochemistry 1996;35:12612–22. [5] Fogac¸a AC, Da Silva PI, Teresa Jr M, Miranda M, Bianchi AG, Miranda A, et al. Antimicrobial activity of a bovine hemoglobin fragment in the tick Boophilus microplus. J Biol Chem 1999;274:25330–4. [6] Froidevaux R, Krier F, Nedjar-Arroume N, Vercaigne-Marko D, Kosciarz E, Ruckebusch C, et al. Antibacterial activity of a pepsinderived bovine hemoglobin fragment. FEBS Lett 2001;491:159–63. [7] Ivanov VT, Karelin AA, Philippova MM, Nazimov IV, Pletnev VZ. Hemoglobin as a source of endogenous bioactive peptides: the concept of tissue-specific peptide pool. Biopoly 1997;43:171–88. [8] Karelin AA, Philippova MM, Ivanov VT. Proteolytic degradation of haemoglobin in erythrocytes leads to biologically active peptides. Peptides 1995;16:693–7. [9] Liepke C, Baxmann S, Heine C, Breithaupt N, St¨andker L, Forssmann WG. Human hemoglobin-derived peptides exhibit antimicro-
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
719
bial activity: a class of host defense peptides. J Chromatogr B 2003;791:345–56. Lignot B, Froidevaux R, Nedjar-Arroume N, Guillochon D. Solvent effect on kinetics of appearance of neokyotorphin VVh4 and a bradykinin-potentiating peptide in the course of peptic hydrolysis of bovine haemoglobin. Biotechnol Appl Biochem 1999;30:201– 7. Lindertrom-lang K. Proteins and enzymes. III. Lane medical lectures, vol. VI. Stanford, CA: Stanford University Press; 1952. p. 53– 72. Nicolas P, Mor A. Peptides as weapons against microorganisms in the chemical defense system of vertebrates. Annu Rev Microbiol 1995;49:277–304. Nyberg F, Sanderson K, Gl¨amsta EL. The hemorphins: a new class of opioid peptides derived from the blood protein haemoglobin. Biopoly 1997;43:147–56. Parish CA, Jiang H, Tokiwa Y, Berova N, Nakanishi K, Mc Cabe D, et al. Broad-spectrum antimicrobial activity of hemoglobin. Bioorg Med Chem 2001;9:377–82. Piot JM, Zhao QY, Guillochon D, Ricart G, Thomas D. Isolation and characterisation of two opioid peptides from a bovine hemoglobin peptic hydrolysate. Biochem Biophys Res Commun 1992;189:101–10. Piot JM, Zhao QY, Guillochon D, Ricart G, Thomas D. Isolation and characterisation of a bradykinin-potentiating peptide from bovine peptic hemoglobine hydrolysate. FEBS Lett 1992;299:75–9. Strub JM, Goumon Y, Lugardon K, Capon C, Lopez M, Moniatte M, et al. Antibacterial activity of glycosylated and phosphorylated chromogranin A-derived peptide 173–194 from bovine adrenal medullary chromaffin granules. J Biol Chem 1996;271:28533–40. Takagi H, Shiomi H, Ueda H, Amano H. A novel analgesic dipeptide from bovine brain is a possible Met-enkephalin realeaser. Nature 1979;5737:410–2. Takagi H, Shiomi H, Hayashi K, Kiso Y, Kitagawa K. Isolation of a novel analgesic pentapeptide, neo-kyotorphin, from bovine brain. Life Sci 1982;31:1733–6. Zhao QY, Piot JM, Gautier V, Cottenceau G. Peptic peptide mapping by HPLC, on line with photodiode array detection, of a haemoglobin hydrolysate produced at pilot-plant scale from an ultrafiltration process. Appl Microbiol Biotechnol 1996;45:778–84. Zhao QY, Sannier F, Piot JM. Kinetics of appearance of four hemorphins from bovine hemoglobin peptic hydrolysates by HPLC coupled with photodiode array detection. Biochem Biophys Acta 1996;1295:73–80. Zhao QY, Molina P, Piot JM. Peptic peptide mapping by HPLC, on line with photodiode array detection, of a haemoglobin hydrolysate produced at pilot-plant scale from an ultrafiltration process. J Liq Chrom Rel Technol 1997;20:1717–39.