Development of continuous type apparatus for ampholyte-free isoelectric focusing (autofocusing) of peptides in protein hydrolysates

An apparatus for continuous fractionation of peptides on the basis of amphoteric nature of sample peptides was developed. A tank (66.5 cm x 8 cm x 8 cm, L x W x H) was divided into 12 compartments by a thin agarose gel layer. A drain tube (5.5 cm in length and 0.7 cm in i.d.) was fixed through the bottom of each compartment to give a height of 4 cm from the bottom. The tank with 12 compartments and electrodes was referred to as an autofocusing unit. The peptide solution or water was delivered to the sample compartments of the first unit. The solutions drained from the first unit were successively delivered to the second and third units. To the electrodes of three units, a direct electric current was applied. By using the present apparatus, peptides in casein hydrolysate can be continuously fractionated at least for 5 h. Better resolution was obtained in the second and third units.     

Isolation and characterization of antioxidative peptides from gelatin hydrolysate of Alaska pollack skin

Gelatin extracted from Alaska pollack skin was hydrolyzed with serial digestions in the order of Alcalase, Pronase E, and collagenase using a three-step recycling membrane reactor. The fraction from the second step, which was hydrolyzed with Pronase E, was composed of peptides ranging from 1.5 to 4.5 kDa and showed high antioxidative activity. Two different peptides showing strong antioxidative activity were isolated from the hydrolysate using consecutive chromatographic methods including gel filtration on a Sephadex G-25 column, ion-exchange chromatography on a SP-Sephadex C-25 column, and high-performance liquid chromatography on an ODS column. The isolated peptides, P1 and P2, were composed of 13 and 16 amino acid residues, respectively; and both peptides contained a Gly residue at the C-terminus and the repeating motif Gly-Pro-Hyp. The antioxidative activities of the purified peptides were measured using the thiobarbituric acid method, and the cell viability was measured with MTT assay. The results showed that P2 had potent antioxidative activity on peroxidation of linoleic acid. Moreover, the cell viability of cultured liver cells was significantly enhanced by addition of the peptide. These results indicate that the purified peptide, P2, from gelatin hydrolysate of Alaska pollack skin is a natural antioxidant which has potent antioxidative activity.     

Human lysozyme possesses novel antimicrobial peptides within its N-terminal domain that target bacterial respiration

Human milk lysozyme is thought to be a key defense factor in protecting the gastrointestinal tract of newborns against bacterial infection. Recently, evidence was found that pepsin, under conditions relevant to the newborn stomach, cleaves chicken lysozyme (cLZ) at specific loops to generate five antimicrobial peptide motifs. This study explores the antimicrobial role of the corresponding peptides of human lysozyme (hLZ), the actual protein in breast milk. Five peptide motifs of hLZ, one helix-loop-helix (HLH), its two helices (H1 and H2), and two helix-sheet motifs, H2-β-strands 1-2 (H2-S12) or H2-β-strands 1-3 (H2-S13), were synthesized and examined for antimicrobial action. The five peptides of hLZ exhibit microbicidal activity to various degrees against several bacterial strains. The HLH peptide and its N-terminal helix (H1) were significantly the most potent bactericidal to Gram-positive and Gram-negative bacteria and the fungus Candida albicans . Outer and inner membrane permeabilization studies, as well as measurements of transmembrane electrochemical potentials, provided evidence that HLH peptide and its N-terminal helix (H1) kill bacteria by crossing the outer membrane of Gram-negative bacteria via self-promoted uptake and are able to dissipate the membrane potential-dependent respiration of Gram-positive bacteria. This finding is the first to describe that hLZ possesses multiple antimicrobial peptide motifs within its N-terminal domain, providing insight into new classes of antibiotic peptides with potential use in the treatment of infectious diseases.     

Antigenotoxic effect of grape seed procyanidin extract in Fao cells submitted to oxidative stress

The protective effects of grape seed procyanidin extract on the repair of H(2)O(2)-induced DNA lesions were tested using Fao cells. Cells were exposed to 600 microM H(2)O(2) for 3 or 21 h. A procyanidin extract from grape seed (PE) was incubated or preincubated (1 h) during the exposure to H(2)O(2). The ability of procyanidins to protect against the genotoxicity of H(2)O(2) was compared with those of the monomeric flavanols (+)-catechin and (-)-epicatechin and the flavonol quercetin. After treatment, DNA damage was monitored using alkaline single-cell gel electrophoresis (the comet assay) (Aherne, S. A.; O'Brien, N. M. Nutr. Cancer 1999, 34, 160-166). At the end of the experiment, PE significantly decreased the damage caused by H(2)O(2). The results also showed that quercetin was the most effective of the flavonoids tested, which is consistent with its powerful antioxidant character. The results indicate that procyanidins are more effective than the corresponding individual monomers, catechin and epicatechin, at preventing DNA lesions in hepatocytes and that this protection is higher after preincubation than after co-incubation.     

Antioxidative stress activity of oligophosphopeptides derived from hen egg yolk phosvitin in Caco-2 cells

The protective effects of hen egg yolk phosvitin phosphopeptides (PPPs) against hydrogen peroxide (H2O2)-induced oxidative stress were evaluated in an in vitro assay using human intestinal epithelial cells. Caco-2 cells were stimulated with 1 mM H2O2 for 6 h, and the secretion of IL-8, a proinflammatory mediator, was determined by ELISA as a biomarker of oxidative stress. The inhibition of H2O2-induced IL-8 secretion from Caco-2 cells was observed by pretreatment for 2 h with PPPs, but not with phosvitin. PPPs also suppressed the formation of malondialdehyde in H2O2-treated Caco-2 cells. Furthermore, intracellular glutathione levels and glutathione reductase activity were elevated by the addition of PPPs. The protective effects of PPPs against H2O2-induced oxidative stress were almost the same as that of glutathione, and PPPs with a high content of phosphorus exhibited higher protective activity than PPPs without phosphorus; however, phosphoserine itself did not show any significant antioxidative stress activity. These findings suggest that oligophosphopeptides from hen egg yolk phosvitin possess novel antioxidative activity against oxidative stress in intestinal epithelial cells and that phosphorus and peptide structure seem to have a key role in the activity.     

Identification of hen egg yolk-derived phosvitin phosphopeptides and their effects on gene expression profiling against oxidative stress-induced Caco-2 cells

Oxidative stress is involved in the initiation and propagation of chronic intestinal pathologies. Bioactive peptides such as egg yolk-derived phosvitin phosphopeptides (PPP3) have been previously shown to reduce in vitro oxidative stress by up-regulating glutathione synthesis and antioxidant enzyme activities. Peptide and gene expression profile analysis of the PPP3 peptides can provide insight into structures involved in signal transduction mechanisms in the antioxidative stress response. The objectives of this research were to identify the PPP3 amino acid sequences before and after simulated gastrointestinal digestion and to assess the genes influenced by PPP3. Peptide sequences were analyzed using ESI Q-TOF-MS/MS, and the expression profile of 84 human oxidative stress and antioxidant defense genes were analyzed. Undigested PPP3 was composed of three main peptides: GTEPDAKTSSSSSSASSTATSSSSSSASSPNRKKPMDE (phosvitin-PV residues 4-41), NSKSSSSSSKSSSSSSRSRSSSKSSSSSSSSSSSSSSKSSSSR (PV residues 155-197), and EDDSSSSSSSSVLSKIWGRHEIYQ (PV residues 244-257) and their fragments. There was limited degradation of PPP3 after gastrointestinal digestion as deduced from the fragment sizes of digested PPP3, which ranged from 5 to 32 amino acids. These fragments were rich in contiguous serines and, in some cases, monoesterified with phosphate. Both undigested and digested PPP3 significantly reduced IL-8 secretion in H(2)O(2)-induced Caco-2 cells, indicating that antioxidative stress bioactivity is retained upon digestion. After PPP3 pretreatment, antioxidant genes associated with oxygen and reactive oxygen species (ROS) metabolism and cellular responses to chemical stimulus, oxidative stress, and ROS are up-regulated in the presence and absence of oxidative stress, thereby contributing to the prevention of intestinal oxidative stress and the promotion of gut health.     

Isolation and characterization of a low molecular weight peptide contained in sourdough

To investigate a sourdough-specific peptide, low molecular weight peptides were extracted from sourdough. The peptide fraction was subjected to two kinds of chromatography to separate the peptides. Reverse-phase chromatography of the peptide fraction in the sourdough showed certain specific peptides. The specific peptide fraction was further separated by gel filtration chromatography. Liquid chromatography tandem mass spectrometry analysis identified one of the peptides as VPFGVG (six-mer). This sequence was estimated to occur at the 287-292 position of a low molecular weight glutenin subunit. The peptide (designed as SDP1) was produced by proteases derived from wheat flour. SDP1 showed angiotensin-converting enzyme (ACE) inhibitory activity, and the 50% inhibitory peptide concentration (IC50) was 336 microM. It is possible that the SDP1 peptide partially confers ACE inhibitory activity in sourdough.     

BF2/肽四聚体的构建及其在特异性T细胞检测上的应用

为构建可溶性鸡 组织相容性复合体(MHC)Ⅰ 类分子（BF2）/肽复合物,大量表达并纯化了可生物素化的MHC Ⅰ类分子重链（BF2-BSP）和轻链（Chβ2m）,并合成了鸡传染性支气管炎病毒（IBV）核蛋白N71~78 T细胞表位肽,将其与纯化后的重组蛋白BF2-BSP和Chβ2m在重折叠缓冲液中复性。复性后在快速蛋白液相色谱仪（FPLC）上分离出复性组装成功的BF2/肽单体复合物。将该单体复合物在体外进行生物素化,其产物再次采用FPLC通过分子筛分离出生物素化后的BF2/肽单体复合物;然后将生物素化后的BF2/肽单体复合物与藻红蛋白（PE）标记的链霉亲和素按一定比例反应生成BF2/肽四聚体。为了检测所制备BF2/肽四聚体是否能应用于检测特异性T细胞,从感染IBV H52株10 d后的SPF鸡分离外周血单核细胞（PBMC）,染色后采用流式细胞术检测,结果表明,所制备的BF2/肽四聚体可用于检测IBV N蛋白特异性T淋巴细胞,检测1周龄SPF鸡接种H52后10 d时针对N蛋白的特异性T细胞比率为3.65%。     

Isolation and characterization of an oxygen radical absorbance activity peptide from defatted peanut meal hydrolysate and its antioxidant properties

Defatted peanut meal hydrolysate (DPMH) was purified using ultrafiltration, gel filtration chromatography, and high-performance liquid chromatography. A tripeptide with strong oxygen radical absorbance capacity (ORAC) was isolated and identified as Tyr-Gly-Ser by ESI-MS/MS. It was then synthesized to measure its antioxidant properties in different systems. The ORAC value of Tyr-Gly-Ser was 3-fold higher than that of glutathione (GSH), and it displayed a stronger protective effect on linoleic acid peroxidation and H(2)O(2)-induced oxidative injury in rat pheochromocytoma line PC12 cells than GSH (p < 0.05). However, Tyr-Gly-Ser showed negligible DPPH radical scavenging activity, reducing power, and no metal chelating ability. The results suggested that Tyr-Gly-Ser displayed antioxidant activity via the hydrogen atom transfer mechanism, and the Tyr at the N-terminal was the hydrogen donor. The ORAC assay was recommended as a reliable and effective method to measure the antioxidant activity in the course of antioxidant peptide isolation.     

Development of a large-scale (50 L) apparatus for ampholyte-free isoelectric focusing (autofocusing) of peptides in enzymatic hydrolysates of food proteins

It has been demonstrated that peptides in enzymatic hydrolysates of proteins can be fractionated on the basis of the amphoteric nature of the sample peptides, by a laboratory-scale isoelectric focusing apparatus, without adding a chemically synthesized carrier ampholyte. This approach is referred to as autofocusing. In the present study, a large-scale (up to 50 L) autofocusing apparatus was developed and tested. A tank (125 cm x 25 cm x 20 cm) was divided into 12 compartments by 11 plates, each with a window covered in a thin agarose gel layer supported by a nylon screen (100 mesh). The compartments at both ends were filled with 0.1 N phosphoric acid (anode) and 0.1 N NaOH (cathode), respectively, functioning as electrode compartments. The remaining compartments were used for sample compartments. Autofocusing was carried out at constant voltage according to two different methods. In method 1, all sample compartments were filled with a 1% water solution of casein or milk whey protein hydrolysates. In method 2, two compartments located in the center of the tank were filled with 5% sample solution and the others were filled with deionized water. Compositional and sequence analyses of the autofocusing fractions revealed that peptides in the two hydrolysates can be fractionated within 24 h by the present apparatus. Better fractionation was obtained by method 2, whereas enrichment of some peptides occurred by using method 1.     

Development of a liquid chromatography-tandem mass spectrometry method using capillary liquid chromatography and nanoelectrospray ionization-quadrupole time-of-flight hybrid mass spectrometer for the detection of milk allergens

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of the tryptic digest of a cleaned-up food matrix extract was used for the detection of milk allergens. The emphasis of this study was on casein, which is the most abundant milk protein and is also considered the most allergenic. A sample cleanup method was developed using an ion exchange column and centriprep device. Cookies spiked with milk powder from 0 to 1250 ppm were extracted, cleaned up, and either digested directly by trypsin or further cleaned up by gel electrophoresis before digestion. The peptide mixture was analyzed on a capillary LC-quadrupole time-of-flight system. Two marker peptides from alphaS1-casein were identified and used for prescreening. The MS/MS data from the mass spectrometry system were processed with Masslynx v4.0 and submitted for database search using either ProteinLynx Global Server or Mascot for protein identification. The LC-MS/MS method, using casein enzyme-linked immunosorbent assay as a reference, was tested on the cookie matrix and was extended to other sample matrices. There were good agreements between the two. This LC-MS/MS method provides a valuable confirmatory method for the presence of casein. It also allows the simultaneous detection of other milk allergens.     

Production and characterization of a single-chain variable fragment linked alkaline phosphatase fusion protein for detection of O,O-diethyl organophosphorus pesticides in a one-step enzyme-linked immunosorbent assay

A single-chain variable fragment (scFv) linked alkaline phosphatase (AP) fusion protein for detection of O,O-diethyl organophosphorus pesticides (O,O-diethyl OPs) was produced and characterized. The scFv gene was prepared by cloning V(L) and V(H) genes from hybridoma cells secreting monoclonal antibody with broad specificity for O,O-diethyl OPs. The amplified V(L) and V(H) regions were assembled using a linker (Gly(4)Ser)(3) by means of splicing overlap extension polymerase chain reaction to obtain the scFv gene, which was cloned into the expression vector pLIP6/GN containing an AP gene to produce the scFv-AP fusion protein in Escherichia coli strain BL21. The protein was purified by antigen-conjugated immunoaffinity chromatography and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and competitive direct enzyme-linked immunosorbent assay (cdELISA). The fusion protein is bifunctional, retaining both antigen binding specificity and AP enzymatic activity. Analysis of spiked and blind river water and Chinese cabbage samples demonstrated that the fusion protein based cdELISA(FP) exhibited good sensitivity and reproducibility.     

Novel, unnatural benzo-1,2,3-thiadiazole-7-carboxylate elicitors of taxoid biosynthesis

In order to establish the chemical biological technology for production of valuable secondary metabolites, a novel family of unnatural elicitors derived from the plant activator benzo-1,2,3-thiadiazole-7-carboxylic acid were designed and synthesized. New synthetic elicitors that showed powerful eliciting activities upon taxoid biosynthesis by Taxus chinensis suspension cells were obtained. For example, benzo-1,2,3-thiadiazole-7-carboxylic acid 2-(2-hydroxybenzoxyl)ethyl ester was more effective and resulted in nearly 40% increase in taxuyunnanine C content and production in comparison with methyl jasmonate, which was previously reported as the most powerful chemical elicitor for taxoid biosynthesis. The novel class of elicitors was found to induce plant defense responses, including promotion of H(2)O(2) levels originating from oxidative burst and activation of phenylalanine ammonia lyase. Interestingly the plant defense responses induced corresponded well to the superior stimulating activity in T. chinensis cell cultures. The work indicates that the newly synthesized benzothiadiazoles can act as a new family of elicitors for taxoid biosynthesis in plant cells.     

Novel approach for large-scale, biocompatible, and low-cost fractionation of peptides in proteolytic digest of food protein based on the amphoteric nature of peptides

A large-scale, biocompatible, and low-cost procedure for peptide fractionation based on the amphoteric nature of peptide is developed. A sample cell (120 x 100 x 50 mm) with four joint tubes (17 mm i.d. and 20 mm in length) on the front and back was prepared. On the end of the joint tubes, a nylon screen (100 mesh)-supported agarose gel layer was formed. Five or nine of the sample cells were connected. A tryptic digest of casein (2.0-3.6 L) was applied to the sample cells. At each end of the sample cell apparatus, an additional cell filled with 0.1 M H(3)PO(4) or NaOH was connected and used as anode and cathode compartments, respectively. Reproducible fractionation of peptide could be achieved by collecting fractions with specific pH values when voltage reached a plateau by applying direct current at constant power. Running time necessary for fractionation of peptide was inversely proportional to electric power and directly proportional to sample volume.     

Modulating effects of thyme and its major ingredients on oxidative DNA damage in human lymphocytes

The present study was carried out to investigate the modulating effects of thyme and its major components against the oxidative DNA damage induced by H(2)O(2). The human lymphocytes with thymol, carvacrol, and gamma-terpinene incubated with or without 0.1 mM H(2)O(2) for 30 min at 37 degrees C and the DNA damage were evaluated by singe cell gel electropheresis (comet assay). Concentrations above 0.1 mM thymol and gamma-terpinene and 0.05 mM carvacrol significantly induced DNA damage in human lymphocytes, but at the smaller concentrations no additional DNA strand breakage has been observed. At the all concentrations studied, gamma-terpinene did not show any protective effect against H(2)O(2) induced oxidative DNA damage, but the phenolic compounds thymol and carvacrol at concentrations below 0.2 and 0.1 mM, respectively, significantly reduced the oxidative DNA damage (p < 0.001). The n-hexane and ethyl acetate fractions prepared from the methanolic extracts of Thymus spicata also were found to inhibit DNA damage.     

Hydrolysis of low-molecular-weight soil peptides by immobilized proteases in column and batch reaction systems

Summary Organic matter was extracted from three soils, a cultivated Berwick sandy loam, a cultivated Franklin loamy sand, and an uncultivated Cumberland silty loam. Gel-permeation chromatography was used to separate organic matter extracts into high- (HMW) and low-molecular-weight (LMW) fractions. Reversed-phase high performance liquid chromatography was used to separate and collect the LMW peptide fractions. Peptide samples were hydrolyzed with immobilized proteases attached to beaded agarose and carboxymethyl cellulose in column and batch reaction systems. The chromatograms suggested that peptides are bound to common soil components. The amino acids released in the greatest percentages were relatively non-polar. Large percentages of serine, glycine, alanine, threonine, and valine were observed in the LMW soil peptides. Little aspartic acid, asparagine, glutamic acid, glutamine, arginine, and no histidine was detected in the LMW soil peptides. The soil peptides released different amino acid percentages and quantities when hydrolyzed by immobilized proteases attached to different supports. The quanitities of amino acids released by batch hydrolysis differed from those obtained with column hydrolysis. Greater quantities of amino acids were released (by both types of immobilized protease) from the LMW peptide hydrolysates of the two cultivated soils than from the uncultivated soil.     

Electron spin resonance estimation of hydroxyl radical scavenging capacity for lipophilic antioxidants

Hydroxyl radical scavenging capacity estimation for lipophilic antioxidants is a challenge due to their poor solubility in aqueous radical generating and measuring systems. In this study, an electron spin resonance (ESR) method was developed and validated for its application in estimating the relative hydroxyl radical (HO*) scavenging capacity for lipophilic antioxidants under physiological pH using a Fenton Fe2+/H2O2 system for radical generation and acetonitrile as a solvent. The Fenton Fe2+/H2O2 system generates a constant flux of pure HO* under the assay conditions. The method was validated by linearity, precision, and reproducibility using selected known lipophilic antioxidants including alpha-tocopherol, lutein, beta-carotene, and BHT. The potential effects of commonly used water-miscible and water-immiscible organic solvents on the Fenton Fe2+/H2O2 HO* generating system as well as their possible interactions with the fluorescent and spectroscopic probes were also reported. In addition, the limitation of the ESR assay was described.     

Immobilization of angiotensin-converting enzyme on glyoxyl-agarose

The assay of angiotensin-converting enzyme (ACE) inhibition by food-derived peptides is usually carried out by using soluble ACE in a batch process. The purification of this enzyme from tissues is not an easy task, and the resulting preparation loses activity very fast. In addition, ACE commercial preparations are very expensive. In this work the immobilization of ACE, through lysine amino groups, to 4% beads cross-linked (4 BCL) glyoxyl-agarose is described. The amount of immobilized enzyme increased with increasing concentrations of enzyme and with incubation time until a saturation point was reached at 50 mg protein/mL gel and 3.5 hours, respectively. The IC50 values for a noncompetitive sunflower peptide inhibitor were similar for the soluble (30.56 microM) and immobilized (32.7 microM) enzymes. An immobilized derivative was obtained that was 60 times more stable than the soluble enzyme at 60 degrees C. This procedure yields a derivative that can be reused and has increased thermal stability compared to that of the soluble enzyme. Thus, ACE immobilization is a good alternative to using soluble freshly prepared or commercial preparations because of economical and practical reasons.     

自由基引起的氧化对牛乳清蛋白凝胶特性的影响

为了深入了解蛋白氧化对凝胶特性的影响,以此探讨乳清蛋白氧化对其功能性质的影响机制,该文主要研究了氧化对乳清蛋白凝胶质地、流变学特性和微观结构变化的影响。试验采用羟基自由基氧化体系,在不同H2O2浓度(1～20mmol/L)及不同FeCl3浓度(0.1～1mmol/L)对乳清蛋白分别氧化3h,通过质构仪、流变仪和扫描电镜对凝胶特性和微观结构进行研究。结果显示:同未氧化乳清蛋白相比,在所有氧化条件下,凝胶硬度降低了90%以上,贮藏模量(G')值降低了17%以上,复合模量(G*)值降低了20%以上;高浓度氧化条件下,弹性降低了20%以上。氧化明显改变了凝胶的微观结构,随着氧化剂的加入,导致了疏松多孔且不规则凝胶的形成。上述结果表明,氧化对蛋白凝胶质地和凝胶形成能力起着很大的破坏作用,并影响着其微观结构。     

Determination of potato glycoalkaloids using a liposome immunomigration, liquid-phase competition immunoassay

Polyclonal anti-solanine antibodies were raised and used in the assembly of a liposome immunomigration, liquid-phase competition strip immunoassay. In this format, a similar cross-reactivity was observed between the glycoalkaloids alpha-solanine and alpha-chaconine. The strip assay was implemented to quantitate total glycoalkaloids (TGA) from potatoes. Recoveries in spiked potato samples using the strip assay and water:acetic acid:sodium bisulfite extracting solvent were in the range of 84-111%. The values of the TGA quantitations by the strip assay as compared with those obtained by HPLC, in nonspiked tubers coming from cloned potato samples donated by potato breeders, were equivalent and highly correlated (r(2) = 0.91). The strip assay proved advantageous over HPLC for extra-laboratory measurements such as the rapid identification of samples that should be rejected due to an elevated TGA content.