Bovine hemoglobin: an attractive source of antibacterial peptides.

A peptic hemoglobin hydrolysate was fractioned by a semi-preparative reversed-phase HPLC and some fractions have an antibacterial activity against four bacteria strains: Micrococcus luteus A270, Listeria innocua, Escherichia coli and Salmonella enteritidis. These fractions were analyzed by ESI/MS and ESI/MS/MS, in order to characterize the peptides in these fractions. Each fraction contains at least three peptides and some fractions contain five peptides. All these fractions were purified several times by HPLC to obtain pure peptides. Thirty antibacterial peptides were identified. From the isolated antibacterial peptides, 24 peptides were derived from the alpha chains of hemoglobin and 6 peptides were derived from the beta chains of hemoglobin. The lowest concentration of these peptides (minimum inhibitory concentration (MIC)) necessary to completely inhibit the growth of four bacteria strain was determined. The cell population of all of the tested bacteria species decreased by at least 97% after a 24-h incubation with any of the peptides at the minimum inhibitory concentration.

Angiotensin I-converting enzyme (ACE) inhibitory activities of sardinelle (Sardinella aurita) by-products protein hydrolysates obtained by treatment with microbial and visceral fish serine proteases.

The angiotensin I-converting enzyme (ACE) inhibitory activities of protein hydrolysates prepared from heads and viscera of sardinelle (Sardinella aurita) by treatment with various proteases were investigated. Protein hydrolysates were obtained by treatment with Alcalase(®), chymotrypsin, crude enzyme preparations from Bacillus licheniformis NH1 and Aspergillus clavatus ES1, and crude enzyme extract from sardine (Sardina pilchardus) viscera. All hydrolysates exhibited inhibitory activity towards ACE. The alkaline protease extract from the viscera of sardine produced hydrolysate with the highest ACE inhibitory activity (63.2±1.5% at 2mg/ml). Further, the degrees of hydrolysis and the inhibitory activities of ACE increased with increasing proteolysis time. The protein hydrolysate generated with alkaline proteases from the viscera of sardine was then fractionated by size exclusion chromatography on a Sephadex G-25 into eight major fractions (P1-P8). Biological functions of all fractions were assayed, and P4 was found to display a high ACE inhibitory activity. The IC50 values for ACE inhibitory activities of sardinelle by-products protein hydrolysates and fraction P4 were 1.2±0.09 and 0.81±0.013mg/ml, respectively. Further, P4 showed resistance to in vitro digestion by gastrointestinal proteases. The amino acid analysis by GC/MS showed that P4 was rich in phenylalanine, arginine, glycine, leucine, methionine, histidine and tyrosine. The added-value of sardinelle by-products may be improved by enzymatic treatment with visceral serine proteases from sardine.

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.

New antibacterial peptide derived from bovine hemoglobin.

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 alpha 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 microM was reported against L. innocua and 76 microM for other various bacterial species. This peptide had no hemolytic activity up to 380 microM concentration.

Iron absorption from concentrated hemoglobin hydrolysate by rat.

Although heme iron is highly bioavailable, the low iron content of hemoglobin prevents its use for dietary fortification; on the other hand, purified heme has low solubility and absorption rate. The present study was designed to assess the interactions between concentrated heme iron and peptides released during globin hydrolysis and cysteine and their relation with iron absorption. Hemoglobin was hydrolyzed by pepsin or subtilisin, and then, heme iron was concentrated by ultrafiltration. Iron absorption was studied in a Ussing chamber; gluconate was used as control. Iron uptake from nonconcentrated pepsin hydrolysate and gluconate was lower than from other groups. Cysteine significantly enhanced iron uptake except from the concentrated subtilisin hydrolysate. There was no significant difference between cysteine-supplemented groups. According to the different hydrolysis pathways of enzymes, it is assumed that the presence of hydrophobic peptides and the strength of heme-peptide interactions are both determining factors of heme iron absorption. These interactions occur mainly before iron uptake, as emphasized by the effect of cysteine.

Nine novel angiotensin I-converting enzyme (ACE) inhibitory peptides from cuttlefish (Sepia officinalis) muscle protein hydrolysates and antihypertensive effect of the potent active peptide in spontaneously hypertensive rats.

This study aimed to identify novel ACE inhibitory peptides from the muscle of cuttlefish. Proteins were hydrolyzed and the hydrolysates were then subjected to various types of chromatography to isolate the active peptides. Nine ACE inhibitory peptides were isolated and their molecular masses and amino acid sequences were determined using ESI-MS and ESI-MS/MS, respectively. The structures of the most potent peptides were identified as Val-Glu-Leu-Tyr-Pro, Ala-Phe-Val-Gly-Tyr-Val-Leu-Pro and Glu-Lys-Ser-Tyr-Glu-Leu-Pro. The first peptide displayed the highest ACE inhibitory activity with an IC50 of 5.22µM. Lineweaver-Burk plots suggest that Val-Glu-Leu-Tyr-Pro acts as a non-competitive inhibitor against ACE. Furthermore, antihypertensive effects in spontaneously hypertensive rats (SHR) also revealed that oral administration of Val-Glu-Leu-Tyr-Pro can decrease systolic blood pressure significantly (p<0.01). These results suggest that the Val-Glu-Leu-Tyr-Pro would be a beneficial ingredient for nutraceuticals and pharmaceuticals acting against hypertension and its related diseases.

Influence of the extent of hemoglobin hydrolysis on the digestive absorption of heme iron. An in vitro study.

This study was designed to assess the interactions of heme with peptides produced by enzyme hydrolysis of hemoglobin, and their relationship with heme iron absorption. Bovine hemoglobin was hydrolyzed by pepsin or by subtilisin, which differ in their hydrolysis processes. The hydrolysis rate ranged from 0 (native hemoglobin) to 15%. Heme solubility and heme-peptides interactions were compared to iron absorption by the Ussing chamber model, at intestinal pH (7.5). Increasing hemoglobin hydrolysis enhanced iron absorption; the highest value was reached between 8 and 11% hydrolysis, whatever the enzyme used. Comparing the products of hydrolysis of the two enzymes showed that heme iron absorption depends not only on its solubility, but relies mainly on the balance between the strength of heme-peptides and the polymerization rate of heme.

Controlled Enzymatic Hydrolysis: A New Strategy for the Discovery of Antimicrobial Peptides.

The use of antimicrobial peptides (AMPs) is an alternative to traditional antibiotics. AMPs are obtained using different methods such as bacterial synthesis, chemical synthesis and controlled enzymatic hydrolysis. The later is an interesting approach that deserves our attention because of the yields gathered and peptides engineered. Usually, activities of AMPs obtained in such a way are tightly dependent on the hydrolysis mechanism used. This paper deals with the hydrolysis of hemoglobin mechanism as a potential source of AMPs. Production of AMPs from hemoglobin using enzymatic controlled system is linked to hemoglobin structure. Further, we show that bovine hemoglobin, which is sensitive to peptic hydrolysis, results upon enzymatic digestion as a great source of AMPs. The hemoglobin in native and denatured states was hydrolyzed by "one-by-one" and "zipper" mechanisms, respectively. Nevertheless, a new mechanism named "semi-zipper" mechanism is obtained when protein is in molten globule structural state, constituting an original strategy for AMPs production. Seventy seven percentage of the peptides obtained by this new strategy showed antibacterial activity against nine strains.

Characterization and identification of a chymotryptic hydrolysate of alpha-lactalbumin stimulating cholecystokinin release in STC-1 cells.

Alpha-lactalbumin hydrolysate is of significant interest, due to its potential application as a source of bioactive peptides in nutraceutical and pharmaceutical domains. This study was focused on the cholecystokinin (CCK) family compounds which are small peptides involved in the satiety control. The action of chymotryptic hydrolysate of alpha-lactalbumin on cholecystokinin release from intestinal endocrine STC-1 cells was investigated. We demonstrated for the first time that a chymotryptic hydrolysate of alpha-lactalbumin was able to highly stimulate CCK-releasing activity from STC-1 cells. The peptidic hydrolysate was characterized by LC/MS and MS/MS, thus highlighting the presence of 11 fractions containing 21 peptides, each potentially having the desired activity.

RYH: a minimal peptidic sequence obtained from beta-chain hemoglobin exhibiting an antimicrobial activity.

Bovine hemoglobin is an animal protein described as source of bioactive peptides. Enzymatic hydrolysis of this protein results into some peptides exhibiting antimicrobial activity against Gram-positive and Gram-negative bacteria. In this study, a family of peptides from the beta chain (beta-114-145 derived peptides) obtained by peptic hydrolysis of bovine hemoglobin, was purified by reverse-phase HPLC and characterized by different analytical techniques (mass spectrometry, circular dichroism). The minimum inhibitory concentration was determined to show the antimicrobial activity of these peptides. Four bacterial strains were used: two Gram-negative (Escherichia coli and Salmonella Enteritidis) and two Gram-positive strains (Listeria innocua and Micrococcus luteus). The effect of these peptides on artificial membrane was also measured. Our findings showed that the peptide ß114-145 and its peptic derivatives contain the RYH sequence. The most antimicrobial peptide is the RYH peptide which was the shortest one.

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.

Obtaining antimicrobial peptides by controlled peptic hydrolysis of bovine hemoglobin.

Under standard conditions, the peptides and specially the active peptides were obtained from either the denatured hemoglobin that all structures are completely modified or either the native hemoglobin where all structures are intact. In these conditions, antibacterial peptides were isolated from a very complex peptidic hydrolysate which contains more than one hundred peptides having various sizes and characteristics, involving a complex purification process. The new hydrolysis conditions were obtained by using 40% methanol, 30% ethanol, 20% propanol or 10% butanol. These conditions, where only the secondary structure of hemoglobin retains intact, were followed in order to enrich the hydrolyzed hemoglobin by active peptides or obtain new antibacterial peptides. In these controlled peptic hydrolysis of hemoglobin, a selective and restrictive hydrolysate contained only 29 peptides was obtained. 26 peptides have an antibacterial activity against Micrococcus luteus, Listeria innocua, and Escherichia coli with MIC from 187.1 to 1 µM. Among these peptides, 13 new antibacterial peptides are obtained only in these new hydrolysis conditions.

Analysis of novel angiotensin I-converting enzyme inhibitory peptides from enzymatic hydrolysates of cuttlefish (Sepia officinalis) muscle proteins.

The angiotensin I-converting enzyme (ACE) inhibitory activities of protein hydrolysates prepared from cuttlefish (Sepia officinalis) proteins by treatment with various bacterial proteases were investigated. The hydrolysate generated by the crude enzyme from Bacillus mojavensis A21 displayed the highest ACE inhibitory activity, and the higher inhibition activity (87.11 +/- 0.92% at 2 mg/mL) was obtained with hydrolysis degree of 16%. This hydrolysate was fractionated by size exclusion chromatography on a Sephadex G-25 into eight major fractions (P(1)-P(8)). Fraction P(6), which exhibited the highest ACE inhibitory activity, was then fractionated by reversed-phase high performance liquid chromatography (RP-HPLC). Eleven ACE inhibitory peptides were isolated, and their molecular masses and amino acids sequences were determined using ESI-MS and ESI-MS/MS, respectively. The structures of the most potent peptides were identified as Ala-His-Ser-Tyr, Gly-Asp-Ala-Pro, Ala-Gly-Ser-Pro and Asp-Phe-Gly. The first peptide displayed the highest ACE inhibitory activity with an IC(50) of 11.6 microM. The results of this study suggest that cuttlefish protein hydrolysates are a good source of ACE inhibitory peptides.

Cathepsin D from the hepatopancreas of the cuttlefish (Sepia officinalis): purification and characterization.

Cathepsin D from the hepatopancreas of cuttlefish ( Sepia officinalis ) was purified to homogeneity by precipitation with ammonium sulfate (30-60%, w/v), Sephadex G-100 gel filtration, Mono-S cation-exchange chromatography, Sephadex G-75 gel filtration, and Mono-S FPLC with a 54-fold increase in specific activity and 17% recovery. The molecular weight of the purified cathepsin D was estimated to be 37.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). On the basis of the native-PAGE and hemoglobin zymography, the purified protease appeared as a single band. The optimum pH and temperature for the cathepsin D activity were pH 3.0 and 50 °C, respectively, using hemoglobin as a substrate. The purified enzyme was completely inhibited by pepstatin A; however, no inhibition was observed with phenylmethylsulfonyl fluoride and ethylenediaminetetraacetic acid. Moreover, the activity was strongly inhibited by SDS and molybdate and enhanced by ATP. The purified cathepsin D was activated by Mg(2+), Ni(2+), Zn(2+), Cu(2+), Cd(2+), Sr(2+), and Co(2+) ions, whereas it was not affected by Na(+), K(+), and Ca(2+) ions. The N-terminal amino acid sequence of the first 13 amino acids of the purified cathepsin D was APTPEPLSNYMDA. S. officinalis cathepsin D, which showed high homology with cathepsin D from marine vertebrates and invertebrates, had a Pro residue at position 6 and a Ser residue at position 8, where Thr and Lys are common in all marine vertebrates cathepsins D. S. officinalis cathepsin D showed high efficiency for the hydrolysis of myofibrillar proteins extracted from cuttlefish muscle.

Ion-pairing separation of bioactive peptides using an aqueous/octan-1-ol micro-extraction system from bovine haemoglobin complex hydrolysates.

The ion-pair concept was applied on complex haemoglobin hydrolysates to extract two opioid peptides, LVV-haemorphin-7 and VV-haemorphin-7, in an aqueous/octan-1-ol micro-extraction system in the presence of alkyl-sulfonic acid as a surfactant agent and in relation to the haemorphin physico-chemical properties (charge, hydrophobicity). The effect of combined alkyl chain length/aqueous phase pH and the haem behaviour during the extraction, on the haemorphin recovery yield and enrichment has been determined. It has proved that transport over the organic phase is mediated by the alkyl-sulfonic acids, whatever be the aqueous phase pH. However, increasing both the alkyl chain length and the pH in the aqueous phase shows an haemorphin enrichment ratio increase but a recovery decrease of the extracted opioid peptides in the organic phase. Therefore, the best conditions to extract LVVh-7 and VVh-7 are the use of the octane-sulfonic acid at aqueous phase pH of 5 or 7 and the octane or the heptane-sulfonic acid with an aqueous phase pH of 5 or 7 respectively. In these conditions, a partition coefficient of 1.64 and 1.60 respectively for LVVh-7 and VVh-7 are obtained and represent about 40 times that acquired without agent.

Covalent attachment of trypsin on plasma polymerized allylamine.

This paper focuses on the immobilization of a proteolytic enzyme, trypsin, on plasma polymerized allylamine (ppAA) films. The later have been deposited onto silicon substrate by means of radiofrequency glow discharge. The covalent attachment of the enzyme was achieved in three steps: (i) activation of the polymer surface with glutaraldehyde (GA) as a linker, (ii) immobilization of trypsin and (iii) imino groups reduction treatment. The effects and efficiency of each step were investigated by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Fluorescent spectroscopy was used to evaluate the change of the biological activity following the immobilization steps. The results showed that enzyme immobilization on GA-modified substrate increases the enzyme activity by 50% comparing to adsorbed enzymes, while the imino reduction treatment improves the enzyme retention by about 30% comparing to untreated samples. In agreement with XPS and AFM data, UV-vis absorption spectroscopy, used to quantify the amount of immobilized enzyme, showed that allylamine plasma polymer presents a high adsorption yield of trypsin. Although the adsorbed enzymes exhibit a lower activity than that measured for enzymes grafted through GA linkers, the highest catalytic activity obtained was for the enzymes that underwent the three steps of the immobilization process.

Cold plasma functionalized TeraHertz BioMEMS for enzyme reaction analysis.

In this paper, we describe the development, functionalization and functionality testing of a TeraHertz (THz) Bio-MicroElectroMechanical System (BioMEMS) dedicated to enzyme reaction analysis. The microdevice was fabricated by mixing clean room microfabrication with cold plasma deposition. The first is used to build the microfluidic circuits and the THz sensor, while the later serves for the polymerization of allylamine using a homemade glow discharge plasma reactor for a subsequent immobilization of enzymatic biocatalysts. Thermal stability of the deposited plasma polymer has been investigated by infrared spectroscopy. Fluorescent detection confirmed the efficiency of the immobilization and the enzyme hydrolysis into the BioMEMS microchannels. For the first time, the progression of the hydrolysis reaction over time was monitored by the THz sensor connected to a vectorial network analyzer. Preliminary results showed that sub-THz transmission measurements are able to discriminate different solid films, various aqueous media and exhibit specific transmission behavior for the enzyme hydrolysis reaction in the spectral range 0.06-0.11 THz.

Continuous production of a peptidic fraction containing the intermediate opioid peptide LVV-haemorphin-7 (LVVh-7) by peptic hydrolysis of bovine haemoglobin in a continuous membrane reactor.

Peptic hydrolysis of native bovine haemoglobin at pH 3 yields the LVV-haemorphin-7 (Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe; LVVh-7) opioid peptide corresponding to the residues-31-40 fragment of the beta-chain of haemoglobin. This peptide is intermediate in the course of batch hydrolysis and is rapidly degraded. Indeed, it shows an optimum at 3% degree of hydrolysis (i.e. 2 min of reaction time). The hydrolysis was carried out in a continuous membrane reactor with a space time (ratio of the flux to the reactor volume) set to 2 min (corresponding to optimum LVVh-7 production). This process allows the continuous production of a constant fraction of intermediate peptides containing LVVh-7 for 48 min.

A kinetic study of bovine haemoglobin hydrolysis by pepsin immobilized on a functionalized alumina to prepare hydrolysates containing bioactive peptides.

The hydrolysis kinetics of native and denatured haemoglobin, using pepsin immobilized on aluminium oxide, was studied in order to produce hydrolysates containing bioactive peptides. Pepsin was immobilized on acidic alumina and on 2-ethanolamine- O -phosphate (2-EAOP)-modified acidic alumina. Surface charge of the two supports was determined as a function of pH. Kinetic studies were performed at 23 degrees C in 0.1 M acetate buffer, pH 4.5. At this pH, the surface charge of the two supports was almost the same. The coating of alumina by 2-EAOP only introduced a two carbon spacer between alumina surface and the reaction medium. Adsorption on the two supports of haemoglobin, haem and peptides produced in the course of hydrolyses were compared. Fixation of 2-EAOP on a pepsin-alumina complex gave hydrolysis kinetics of urea-denatured haemoglobin close to that obtained with the same amount of pepsin in solution, but with comparatively less adsorption of peptides and complete adsorption of haem. Heterogeneous hydrolyses of haemoglobin with pepsin, immobilized on functionalized alumina, resulted in the presence of VV-haemorphin-4, VV-haemorphin-7 and neokyotorphin in the supernatants without haem, the presence of which makes further purification difficult.

Kinetic study of the appearance of an anti-bacterial peptide in the course of bovine haemoglobin peptic hydrolysis.

The kinetics of the alpha (1-23) peptide, which is the first anti-bacterial peptide to be isolated from a haemoglobin hydrolysate, was studied in the course of peptic hydrolysis at pH 4.5 and 23 degrees C in an homogeneous-phase system. A one-step reversed-phase HPLC coupled with photodiode array detector method was applied to identify and isolate this anti-bacterial peptide. The kinetics of peptide appearance were investigated in acetate buffer alone and in urea as a haemoglobin-denaturing agent. Two different mechanisms, 'one-by-one' for native haemoglobin hydrolysis and 'zipper' for denatured haemoglobin hydrolysis, were observed. Whatever the haemoglobin state, native or denatured, and whatever the hydrolytic mechanism, one-by-one or zipper, the anti-bacterial alpha (1-23) peptide is a transient peptide. To prepare the alpha (1-23) peptide it is suitable to hydrolyse haemoglobin in the presence of urea at a corrected degree of hydrolysis (DH(c)) of 13.5%. The amount of peptide produced in the presence of urea was twice as high as for the hydrolysis of native haemoglobin. The yields of alpha (1-23) peptide with respect to haemoglobin at the optimal DH(c) values were 55 and 25% respectively.