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To address the complexity of the proteome in mass spectrometry (MS)-based top-down proteomics, multidimensional liquid chromatography (MDLC) strategies that can effectively separate proteins with high resolution and automation are highly desirable. Although various MDLC methods that can effectively separate peptides from protein digests exist, very few MDLC strategies, primarily consisting of 2DLC, are available for intact protein separation, which is insufficient to address the complexity of the proteome. We recently demonstrated that hydrophobic interaction chromatography (HIC) utilizing a MS-compatible salt can provide high resolution separation of intact proteins for top-down proteomics. Herein, we have developed a novel 3DLC strategy by coupling HIC with ion exchange chromatography (IEC) and reverse phase chromatography (RPC) for intact protein separation. We demonstrated that a 3D (IEC-HIC-RPC) approach greatly outperformed the conventional 2D IEC-RPC approach. For the same IEC fraction (out of 35 fractions) from a crude HEK 293 cell lysate, a total of 640 proteins were identified in the 3D approach (corresponding to 201 nonredundant proteins) as compared to 47 in the 2D approach, whereas simply prolonging the gradients in RPC in the 2D approach only led to minimal improvement in protein separation and identifications. Therefore, this novel 3DLC method has great potential for effective separation of intact proteins to achieve deep proteome coverage in top-down proteomics. [ABSTRACT FROM AUTHOR]

• Reverse phase column for SEC and RP separation of hydrophilic and hydrophobic macro molecules. • Use of phosphate buffer for sensitive detection of polysaccharide at UV 200 nm. • SEC-RPLC/UV method is simple and generic for analysis of free polysaccharides. The level of free polysaccharide is a critical quality attribute of polysaccharide-protein conjugate vaccines. The work presented describes a simple and sensitive method for the determination of low level free polysaccharides in multiple polysaccharide-protein conjugates. The method utilizes a reverse phase (RP) column to perform a size exclusion chromatography (SEC) separation of free polysaccharide and a reverse phase liquid chromatography (RPLC) separation of free protein and protein-polysaccharide conjugate. The use of phosphate buffer in the mobile phase enables the universal and sensitive detection of low level free polysaccharides at UV 200 nm. The method has been validated to monitor low level free polysaccharide (<1 %) in multiple polysaccharide-protein conjugates. The limit of quantitation is 2 μg/ml or 0.3 % free polysaccharide in 0.6 mg/ml polysaccharide-protein conjugate. The accuracy is in the range of 94.1.0–108.5 %. [ABSTRACT FROM AUTHOR]

Small-aroma chicken with a high protein content, is widely distributed in the mountainous area of Yunnan-Guizhou Plateau, China. In this work, three novel ACE-inhibitory peptides, Leu-Thr-Glu-Lys-Val-Val-Phe (LTQKVVF), Leu-Asp-Asp-His-Phe-Leu (LDDHFL), and Val-Pro-Gly-Pro-Glu-Pro-Lys-Pro (VPGPEPKP), were prepared from the Small-Aroma Chicken breast. The purification and identification protocol employed comprised ion-exchange chromatography, gel filtration chromatography, reverse phase-high performance liquid chromatography (RP-HPLC), and nano liquid chromatography-electrospray ionization–tandem mass (Nano-LC-ESI–MS/MS). Compared with VPGPEPKP and VPGPEPKP, LTQKVVF displayed noticeable ACE inhibitory activity, with IC 50 values of 189.34 μM. What's more, the Lineweaver-Burk plots revealed peptides LTQKVVF and LDDHFL acted as the mixed-competitive inhibitor while VPGPEPKP inhibited ACE in a manner similar to uncompetitive inhibition. The binding free energies of LTQKVVF, LDDHFL, and VPGPEPKP for ACE were −8.1 kcal/mol, −8.6 kcal/mol, and −9.6 kcal/mol, respectively. The strong inhibition of ACE by VPGPEPKP may be attributed to the interaction of hydrogen bonds, alkyl, and pi-alkyl, which formed by crucial residues of ACE. [Display omitted] • Three novel peptides were characterized from small-aroma chicken. • The peptides exhibited inhibition activity on angiotensin converting enzyme (ACE). • Lineweaver Burk plots was used to identify the inhibition mode of peptides. • The interactions between peptides and ACE were simulated by molecular docking. [ABSTRACT FROM AUTHOR]

[Display omitted] • A simple method for the quantitative analysis of purines in beer has been developed. • The SERS probe prepared using a simple electrochemical treatment method on a Ag wire, exhibits a Raman enhancement factor of ∼106. • A linear relationship between SERS intensity and purine concentration in the range of 10−5 to 10−1 mg/mL was obtained for quantification of purines. • The recovery rate of each purine base using the SERS probe exceeds 80% for market beer. The common quantification methods of purines, including high-performance liquid chromatography, micellar electrokinetic chromatography, reverse phase ion pair chromatography, and capillary zone electrophoresis, have been used for the analysis of purines. These conventional techniques are limited by long operation times, large sample volumes, requirements for pretreatment and separation processes, or sophisticated, bulky and expensive equipment. Here, we have developed a simple method to prepare a probe for the quantitative analysis of purines using the surface-enhanced Raman scattering (SERS) method. The SERS probe is a rough Ag wire obtained with simple electrochemical treatment, which is optimized to reach the maximum SERS signal enhancement. The Raman enhancement factor (EF) of Ag wire after roughening can reach ∼106. Under optimized conditions, the rough Ag wire is capable of recognizing four purine bases (namely, guanine, adenine, hypoxanthine, and xanthine) in the range of 10−5 to 10−1 mg/mL with a linear relationship between SERS intensity of signature peak and purine concentration. The recovery rate of each purine base is higher than 80% in the mixture of four purines. The overall method was successfully applied to the monitoring of the level of purines in marketplace beer beverages. The novel wire-based SERS sensor used with portable Raman spectroscopy is prospective in diet safety and personalized point-of-care medical detection. [ABSTRACT FROM AUTHOR]

HPLC, also known as High Performance Liquid Chromatography is a technique used to separate individual components in a single mixture. There are various types of this technique. They are partition chromatography, Normal phase chromatography, Reverse phase chromatography, size exclusion chromatography, ion exchange chromatography, bioaffinity chromatography and aqueous chromatography. The most popular one is the reverse phase chromatography. The mechanism of high performance liquid chromatography relies on pumps in which the solvent which is the pressurized liquid is passed through a solid adsorbent material. In this review, around 50 articles from Pubmed, Google scholar and other article databases which are relevant to the topic are taken. The types, parameters, instrumentation, major and other applications of HPLC are discussed. The various applications of high performance liquid chromatography were discussed and compared with the current and existing articles. The opposing and limiting factors are also discussed. High performance liquid chromatography is a reliable method used as a confirmatory test accordingly. HPLC in combination with other methods produce a very accurate result without any flaws. Many studies have been going on to analyse and reduce the flaws to produce a better insight over the usage of this method. [ABSTRACT FROM AUTHOR]

Umami‐ and kokumi‐taste‐active peptides in Maillard reaction products of enzymatic hydrolysate of bovine bone marrow extract (BBME) were purified and identified by ultrafiltration, gel filtration chromatography, reverse‐phase high‐performance liquid chromatography, liquid chromatography/quadrupole‐time of flight mass spectrometry (LC‐Q‐TOF‐MS/MS) and sensory evaluation. Eight umami and kokumi taste oligopeptides were found in the enzymatic hydrolysate of BBME. After Maillard reaction, the numbers of oligopeptides increased to twelve, such as Ala‐His, Ala‐Val‐His, Gly‐Pro, His‐Gly, Pro‐Ala‐His, Pro‐Val‐Asn‐His, Phe‐Glu‐Ala, Ala‐Ala‐Cys‐Arg, Leu‐Met, Leu‐Thr‐His, Pro‐Gly‐His, Ser‐Thr‐Gly‐Arg, which are linked to a variety of functional properties, such as ACE inhibitor, prolyl endopeptidase inhibitor and dipeptidyl peptidase IV inhibitor, etc. Eight peptides, including Cys‐Pro‐Arg, Ser‐Gly‐Val‐Glu, Pro‐Cys, Cys‐Glu, Cys‐Met‐Thr, Phe‐Glu‐Ala, Ala‐Gln and Leu‐Met, were synthesised and then subjected to sensory evaluation. The evaluation showed that among the eight peptides, Cys‐Pro‐Arg, Pro‐Cys and Leu‐Met led to the distinctly enhanced kokumi taste in blank beef soup. The influence of enzymatic hydrolysis and Maillard reaction to taste‐active components in bovine bone extract including amino acids, nucleotide and peptides. [ABSTRACT FROM AUTHOR]

The mechanism of action of food-derived angiotensin-I-converting enzyme (ACE) inhibitory peptides has not been completely elucidated. In the present study, ion-exchange chromatography, gel filtration chromatography, reverse phase-high performance liquid chromatography, and liquid chromatography-electrospray ionization–tandem mass (LC-ESI-MS/MS) were employed for purifying and identifying the ACE inhibitory peptides from hazelnut. To understand the mode of action of these peptides, ACE inhibition kinetics, in vitro and in vivo bioavailability assays, active site analysis, and interaction between the inhibitory peptides and ACE were investigated. The results identified novel ACE inhibitory peptides Ala-Val-Lys-Val-Leu (AVKVL), Tyr-Leu-Val-Arg (YLVR), and Thr-Leu-Val-Gly-Arg (TLVGR) with IC 50 values of 73.06, 15.42, and 249.3 μM, respectively. All peptides inhibited the ACE activity via a non-competitive mode. The binding free energies of AVKVL, YLVR, and TLVGR for ACE were −3.46, −6.48, and −7.37 kcal/mol, respectively. The strong inhibition of ACE by YLVR may be attributed to the formation of cation–pi interactions. [ABSTRACT FROM AUTHOR]

Aconitum karacolicum from northern Kyrgyzstan (Alatau area) contains about 0.8–1% aconitine as well as other aconite derivatives that have already been identified. In this paper, we compare several methods for the further purification of an Aconitum karacolicum extract initially containing 80% of aconitine. Reverse-phase flash chromatography, reverse-phase semi-preparative HPLC, centrifugal partition chromatography (CPC) and recrystallization techniques were evaluated regarding first their efficiency to get the highest purity of aconitine (over 96%) and secondly their applicability in a semi-industrial scale purification process (in our case, 150 g of plant extract). Even if the CPC technique shows the highest purification yield (63%), the recrystallization remains the method of choice to purify a large amount of aconitine as i) it can be easily carried out in safe conditions; ii) an aprotic solvent is used, avoiding aconitine degradation. Moreover, this study led us to the identification of lappaconitine in Aconitum karacolicum , a well-known alkaloid never found in this Aconitum species. [ABSTRACT FROM AUTHOR]

Objectives. To evaluate the healing effect of the hydroalcoholic extract of Piper aduncum on cell line Human Dermal Fibroblast Adults (hDFa). Materials and methods. The extract was obteined by solid-liquid extraction, then concentrated and lyophilized. The proteins contained in the hydroalcoholic extract was purified; by the high-performance liquid chromatography Reverse Phase RP-HPLC; protein peaks from the HPLC purification via RP- were identified by tandem mass spectrometry of tryptic peptides and analyzed by MALDI-TOF-TOF Mass Spectrometer on ABSciex 4800 (Applied Biosystems); these N-terminal sequences of the 5 purified proteins, which were called K1 K2; K3; K4 and K5, were identified. The values of mean effective concentration (EC50), mean inhibitory concentration (IC50), and the percentage of cell proliferation were determined by MTT assays. Cell migration was assessed by scratch. Also the expression of growth factors was analyzed: platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and Epidermal Growth Factor (EGF), using the technique of polymerase chain reaction with real-time Reverse Transcriptase (RT-qPCR). Results. The hydroholic extract showed an IC50 of 200µg/ mL and the EC 50 value was 103.5 mg/mL in HDFA cell line. Cell proliferation assay showed that protein K2 (1ug/mL) increased proliferation compared to other treatments. In the fibroblast migration assay, K2 (50ug/mL) protein showed increased activity. The relative expression of platelet-derived growth factor (PDGF) increased 8.6 folds compared to the control in the presence of K2 protein. Conclusions. The hydrocoholic extract of Piper aduncum, as well as isolated proteins, increased proliferation and migration of fibroblasts (HDFA); likewise they increased the expression of growth factors involved in the healing process. In wound model in rats the angiogenesis, re-epithelialization and fibroplasia were stimulated. [ABSTRACT FROM AUTHOR]

Through Sephadex gel chromatography, reverse-phase-high-performance-liquid-chromatography and ultra-high performance-liquid-chromatography coupled with electrospray ionization mass spectrometry analysis, a novel tetrapeptide (Ser-Glu-Leu-Glu) of excellent ferrous-chelating capacity (41.24 μmol/g) was identified in millet bran globulin hydrolysate. Physicochemical properties and security of SELE were predicted in silico. The result showed that SELE was no toxic. Based on a model generated from response surface methodology, the optimum preparation conditions of SELE-ferrous chelate were obtained with the mass ratio of SELE to FeCl 2 ·4H 2 O of 43.3: 1, at pH 6.7 and 28.7 °C for 28.8 min. The ferrous-chelating ratio of SELE was 97.06% under these conditions. Structure of SELE-ferrous chelate was characterized with ultraviolet spectroscopy, Fourier-infrared spectroscopy and scanning electron microscope. The results demonstrated that amino group and carboxyl group of SELE were the primary ferrous-chelating sites. Moreover, SELE-ferrous chelate showed higher iron-solubility than FeCl 2 and ferrous lactate against different pasteurization conditions, heating at 100 °C for 30 min, and various pH values (P < 0.05). SELE-ferrous chelate also demonstrated higher iron-solubility than FeCl 2 and ferrous lactate against simulated gastrointestinal digestion (P < 0.05). These results indicated that SELE could be used as ingredient of iron fortifications and had potential applications in the food industry. [Display omitted] • SELE of high Fe2+ chelating ability was identified in millet globulin hydrolysates. • Amino group and carboxyl group of SELE were the primary ferrous chelating sites. • Iron solubility of SELE-Fe2+ chelate is stable under most pasteurization conditions. • SELE-Fe2+ chelate is more stable than FeCl 2 at various pH (P < 0.05). • SELE-Fe2+ chelate showed the highest stability against gastrointestinal digestion. [ABSTRACT FROM AUTHOR]

• Relayed Chromatography was proposed and fulfilled by in situ concentration. • Combination of different chromatography with retained resolution was realized. • This strategy largely shorten the time for searching optimal solvent system. • This strategy allows CCC to achieve better separation in a non-optimal condition. • Eight natural naphthaquinones involving a new compound were isolated. Chromatography is an essential method for separating natural products. In this study, we proposed the concept of 'relayed chromatography', based on the strategy of combining different chromatography with relayed resolution by in-situ concentration technique. The following chromatographic methods were used: high-speed countercurrent chromatography (HSCCC), silica gel liquid chromatography (silica gel LC), and reverse phase liquid chromatography (reverse phase LC). The proposed strategy was effectively applied to the preparative separation of naturally existing naphthaquinones. After the first separation stage (silica gel LC), acetylalkannin (1) was directly collected, while fractions 1, 4 and 5 were collected and respectively subjected to recycling CCC separation after concentration. Thus, deoxyshikonin (2), 8- O -methyl-11- O -acetylshikonin (6), β -acetoxyisovalerylalkannin (7) and alkannin (8) were collected. Fraction 2 was concentrated and injected in reverse phase LC separation. After collection of isobutyrylalkannin (3), the remaining effluent from reverse phase LC retained the peak resolution (R 4,5 =0.45) and was injected into a recycling CCC elution. Finally, β, β -dimethylacrylalkannin (4), and isovalerylalkannin (5) were collected with sufficient resolution (R 4,5 =1.25). Eight naturally occurring naphthaquinones were thus isolated from Arnebia euchroma. The purities of all the compounds were determined by HPLC to be > 90%, and the chemical structures were determined by spectral method. Among the aforementioned compounds, 8- O -methyl-11- O -acetylshikonin (6) was separated as a new compound from A. euchroma. In conclusion, the relayed strategy that retains the resolution of the previous chromatographic stage can improve CCC separation efficiency, which may expand the range of application of CCC combined with different chromatography to the separation of natural products. [ABSTRACT FROM AUTHOR]

Angiotensin-I-converting enzyme (ACE) inhibitory peptides were isolated from walnut pepsin hydrolysates using ultrafiltration, Sephadex G-25 chromatography, reverse-phase high-performance liquid chromatography (RP-HPLC), and the ÄKTA preparative HPLC separation and purification system. ACE inhibitory activity was measured using in vitro HPLC analysis, and N-terminal amino acid sequencing was used for structural identification. Those fractions with the highest ACE inhibitory activity were further purified using RP-HPLC and the ÄKTA systems. The most active ACE inhibitory peptide had the sequence Tyr-Val-Pro-His-Trp-Asp-Leu and a molecular weight of 929 Da. Its activity, along with the activity of a synthetic ACE inhibitory peptide with the same sequence, was measured using HPLC following in vitro gastrointestinal digestion experiments. The IC values obtained before and after digestion were 0.136 and 0.173 μM/mL, respectively, which indicated that the peptide was relative stable during digestion and might have in vivo biological activity. [ABSTRACT FROM AUTHOR]

Protease inhibitors (PI) discovered to be widely distributed in animals, plants and microorganisms, but the information on bacterial PI is scarce. Trypsin inhibitor, named SMTI, was purified from local actinomycete strain (Kuala Lumpur, Malaysia), Streptomyces misionensis UMS1, by acetone precipitation and trypsin-agarose affinity chromatography. Reverse-phase HPLC did not reveal isoinhibitor in purified PI. The purification resulted in a 14 kDa protein, visualized on SDS-PAGE and analyzed with MALDITOF/ TOF. Isoelectric focusing of SMTI demonstrated a single protein with an acidic pI of 6.2. SMTI showed inhibitory activity towards trypsin (74%) and chymotrypsin (41%). Kinetic analysis of trypsin inhibitory activity of SMTI revealed a competitive mechanism with an inhibition constant, Ki of 5 × 10-7 M. SMTI was also shown to have a significant ability to enhance the acitivity of α-amaylase (47%). Furthermore, SMTI exhibited an antibacterial activity against Bacillus cereus, Erwinia, Ralstonia, Salmonella typhi, and Escherichia coli with MIC of 0.06, 0.06, 0.015, 0.12, and 0.48 mg/mL, respectively [ABSTRACT FROM AUTHOR]

Our knowledge of the biological and environmental reactivity of siderophores is limited by the difficulty and cost of obtaining reasonable quantities by purification or synthesis. In this note, we describe a modified procedure for the low-cost, mg-scale purification of pyoverdin-type siderophores using a dual-flash chromatography (reverse-phase absorption and size exclusion) approach. [ABSTRACT FROM AUTHOR]

Abstract: The proteome of the venom of Naja haje legionis, the only medically important elapid species in Morocco, has been elucidated by using a combination of proteomic techniques that includes size exclusion chromatography, reverse-phase HPLC, Tricine/SDS-Page, tryptic digestion, Q-TOF tandem mass spectrometry and database search. The sequence analysis of venom fractions revealed a highly complex venom proteome which counts a total of 76 proteins identified from database that can be assigned into 9 proteins families. We report the identification of: cobra venom factor (CVF), l-amino-acid oxidases (LAAO), acetylcholinesterase (AChE), snake venom metalloproteinases (SVMP), cysteine rich secretory proteins (CRISP), venom nerve growth factor (vNGF), phospholipases A2 (PLA2), vespryns, kunitz-type inhibitor, short neurotoxins, long neurotoxins, weak neurotoxins, neurotoxin like proteins, muscarinic toxins, cardiotoxins and cytotoxins. Comparison of these proteins showed high sequence homology with proteins from other African and Asian cobras. Further works are needed to assess the contribution of individual toxins in venom toxicity. Biological significance: Naja haje legionis is one of the medically important snakes implicated in the pathogenesis of snake bite in Morocco. The absence of information about venom composition and clinical manifestations of envenomation by this cobra represents an obstacle for the management of this environmental disease in the country. The elucidation of Moroccan cobra venom composition will provide a reasonable guidance for clinician to understand the pathophysiological conditions associated with cobra envenomation and the elaboration of better management strategies. [Copyright &y& Elsevier]

Abstract: In the present work, we investigated the structural modifications occurring during the dry heating of model whey proteins, β-lactoglobulin and α-lactalbumin. Samples were adjusted to pH 6.5, water activity aw =0.23 and dry heated at 100°C for up to 24h, and the structural modifications followed by gel permeation chromatography, reverse phase-HPLC, SDS PAGE and mass spectrometry (LC–MS/MS). The dry heating treatment traps a fraction of the proteins into covalently linked soluble aggregates. Moreover, a high proportion of non-aggregated α-lactalbumin (about 73%) was converted into non-native forms. The characteristic of those non-native species was the loss of one or two water molecules per α-lactalbumin molecules. Using tandem mass spectrometric peptide mapping, these chemical modifications were found to be attributed to (i) the formation of a pyroglutamic acid from the N-terminal glutamic acid and (ii) the formation of an internal cyclic imide at position Asp64. The non-native species were not favored in the case of β-lactoglobulin as they represented less than 18% of non-aggregated proteins. [Copyright &y& Elsevier]

Bleu TX-III was isolated from Bothrops leucurus snake venom on one-step analytical chromatography reverse phase HPLC, was homogeneous on SDS-PAGE, and was confirmed by Q-Tof Ultima API ESI/MS (TOF MS mode) mass spectrometry in 14243.8 Da. Multiple alignments of Bleu TX-III show high degree of homology with basic PLA2 myotoxins from other Bothrops venoms. Our studies on local and systemic myotoxicity "in vivo" reveal that Bleu TX-III is myotoxin with local but not systemic action due to the decrease in the plasmatic CK levels when Bleu TX-III is administrated by intravenous route in mice (dose 1 and 5 g). And at a dose of 20 μg myotoxin behaves like a local and systemic action. Bleu TX-III induced moderate marked paw edema, evidencing the local increase in vascular permeability. The inflammatory events induced in the mice (I.M.) were investigated. The increase in the levels of IL-1, IL-6, and TNF-α was observed in the plasma. It is concluded that Bleu TX-III induces inflammatory events in this model. The enzymatic phospholipid hydrolysis may be relevant to these phenomena. Bothrops leucurus venom is still not extensively explored, and the knowledge of its toxins separately through the study of structure/function will contribute for a better understanding of its action mechanism. [ABSTRACT FROM AUTHOR]

Abstract: This paper reports the biochemical and pharmacological characterization of a new myotoxic PLA2 (EC 3.1.1.4) called PhTX-I, purified from Porthidium hyoprora venom by one step analytical chromatography reverse phase HPLC. The homogeneity of the PhTX-I fraction and its molecular mass were initially evaluated by SDS–PAGE and confirmed by MALDI-TOF spectrometry, indicating a molecular mass of 14.249Da and constituted of a single polipeptidic chain. Amino acid sequence was determined by “de novo sequencing,” in tandem mass spectrometry, belonging to D49-PLA2 enzyme class and exhibiting high identity (44–90%) with other myotoxics PLA2 from snake venoms. The enzymatic investigation showed maximal activity at pH 8 and 35–45°C. This activity was dependent on Ca2+, other cations (Mg2+, Mn2+, Cd2+ and Zn2+) reduced notably the enzymatic activity, suggesting that the arrangement of the catalytic site presents an exclusive structure for Ca2+. Ex vivo, whole venom and PhTX-I PLA2 caused blockade of the neuromuscular transmission in young chick biventer cervicis preparations similar to other isolated snake venom toxins from the Bothrops genus. In vivo, both induced local myotoxicity and systemic interleukin-6 response upon intramuscular injection, additionally, induced moderate footpad edema. In vitro, both induced low cytotoxicity in skeletal muscle myoblasts, however PhTX-I PLA2 was able to lyse myotubes. [Copyright &y& Elsevier]

Hydrophobicity is the main determinant in the partitioning of proteins in Aqueous Two-Phase Systems (ATPSs). There are a range of different methods available to evaluate hydrophobicity, including hydrophobic interaction chromatography, reverse phase chromatography, precipitation with ammonium sulphate, average surface hydrophobicity (3-D structure), aminoacid (AA) composition, hydrophobic imbalance, and a statistical description of the AA distribution. This paper reviews the present state of the art in the practical and theoretical evaluation and measurement of a protein's hydrophobicity and evaluates which are the best methodologies to estimate hydrophobicity to predict behaviour in ATPSs. The two methods that are more attractive, depending on the ATPS used, are those that consider the 3-D structure and the hydrophobicity of AA on the surface and the one that uses the parameter 1/m*, which is very simple to determine experimentally. [ABSTRACT FROM AUTHOR]

Abstract: This work reports the purification, biological characterization and amino acid sequence of two new basic PLA2 isoforms, Cdc-9 and Cdc-10, purified from the Crotalus durissus cumanensis venom by one step analytical chromatography reverse phase HPLC. The molecular masses of the PLA2 were 14,175±2.7 Da for Cdc-9 and 14,228±3.5 Da for Cdc-10 both deduced by primary structure and confirmed by MALDI-TOF. The isoforms presented an amino acid sequence of 122 amino acid residues, being Cdc-9: SLVQFNKMIK FETRKSGLPF YAAYGCYCGW GGQRPKDATD RCCFVHDCCY GKVAKCNTKW DIYSYSLKSG YITCGKGTWC KEQICECDRV AAECLRRSLS TYKNEYMFYP DSRCREPPEY TC with pI value of 8.25 and Cdc-10: SLLQFNKMIK FETRKSGVPF YAAYGCYCGW GGRRPKDPTD RCCFVHDCCY GKLTKCNTKW DIYSYSLKSG YITCGKGTWC KEQICECDRV AAECLRRSLN TYKNEYMFYP DSRCRGPPEY TC with a pI value of 8.46, showing highly conserved Ca2+-binding and catalytic sites. The PLA2 activity decreased when the isoforms Cdc-9 and Cdc-10 were incubated with 4-bromophenacyl bromide (p-BPB), anhydrous acetic acid and p-nitrobenzene sulfonyl fluoride (NBSF) when compared with the activity of both native isoforms. In mice, the PLA2 isoforms Cdc-9 and Cdc-10 induced myonecrosis and edema. Myotoxic and edema activities were reduced after treatment of the isoforms with p-BPB; acetylation of the lysine residues and the treatment of PLA2 with NBSF have also induced edema reduction. However, p-BPB strongly diminishes the local and systemic myotoxic effects. [Copyright &y& Elsevier]

OBJECTIVE:: The physicochemical approach, described by Stewart to investigate the acid-base balance, includes the strong ion gap (SIG), a quantitative measure of 'unmeasured' anions, which strongly correlates to the corrected anion gap. The chemical nature of these anions is for the most part unknown. We hypothesized that amino acids, uric acid, and organic acids could contribute to the SIG. DESIGN:: Prospective observational study. SETTING:: Intensive care department of an academic hospital. PATIENTS:: Consecutive intensive care unit patients (n = 31) with metabolic acidosis, defined as a pH of <7.35 and a base excess of /=5 mEq/L. 'Unmeasured' anions were examined by ion-exchange column chromatography, reverse-phase high-performance liquid chromatography, and gas chromatography/mass spectrometry measuring amino acids, uric acid, and organic acids, respectively. MAIN RESULTS:: Comparison of patient characteristics of both SIG groups showed that age, sex, Acute Physiology and Chronic Health Evaluation II, pH, base excess, and lactate were not significantly different. Renal insufficiency and sepsis were more prevalent in the SIG >/=5 mEq/L group (n = 12; median SIG, 8.3 mEq/L), associated with higher mortality. Concentrations of the anionic compounds aspartic acid, uric acid, succinic acid, pyroglutamic acid, p-hydroxyphenyllactic acid, and the semiquantified organic acid homovanillic acid were all statistically significantly elevated in the SIG >/=5 mEq/L group compared with the SIG

The detection of antibodies against the transmembrane glycoprotein (gp21) is very important for the diagnosis of Human T-Cell Leukemia Virus (HTLV) infection, because it has been demonstrated that this protein is a predominant viral antigen, the one that stimulates the antibody production in HTLV-infected individuals. The recombinant proteins and the synthetic peptides corresponding to this glycoprotein are broadly utilized, as antigens, in the HTLV immunodiagnosis. The aim of this work was to obtain, by means of chemical synthesis in solid phase, two peptides: gp21 (13) (361-404) and gp21 (M-1) (377-400) of the gp21 HTLV-I transmembrane protein. The yield obtained in the gp21 (M-1) peptide synthesis was 85 % and for gp21 (13) peptide was 80 %. The two peptides were purified by High Performance Liquid Chromatography Reverse-Phase, and characterized by Electrospray Ionization Mass Spectrometry. Both synthetic peptides were evaluated in an UltramicroELISA assay, with characterized HTLV-I-positive samples, obtaining a reactivity of 92 and 62 % for peptides gp21 (13) and gp21 (M-1), respectively. The specificity for the two synthetic peptides was 100 %. This study demonstrated the possibility to obtain peptides of gp21 protein, through chemical synthesis in solid phase, and their utility as antigens in the HTLV immunodiagnosis. [ABSTRACT FROM AUTHOR]

Abstract: The peptide composition of a cheese reflects its characteristic ripening process, and in this study, large hydrophobic phosphopeptides that accumulate in semi-hard cheese, Herrgård, were identified. Anion exchange chromatography, reverse phase (RP) HPLC, liquid chromatography mass spectrometry and N-terminal amino acid sequencing were used. Milk from homozygotic cows was used to prepare β-casein A1 and A2, and peptides produced from their hydrolysis by plasmin were analysed to support identification of the peptides in cheese. Eight large phosphopeptides released by plasmin hydrolysis of β-casein were identified in the semi-hard cheese, i.e., fractions (f29–105, f29–107, f1–105, f1–107) A1 and A2. Four other peptides that accumulated in the cheese, co-eluted on RP-HPLC with the large primary plasmin-derived phosphopeptides and contributed significantly to two characteristic large peaks, i.e., one peak for each of the two genetic variants A1 and A2 of two β-casein fractions, mainly (f29–93) but also (f30–93). [Copyright &y& Elsevier]

A soy protein hydrolysate was found to inhibit rat platelet aggregation induced by ADP, an aggregating agent. To find out its principal antiplatelet peptide(s), the soy protein hydrolysate was separated successively by gel filtration chromatography, reverse-phase HPLC, and cation exchange HPLC. During the course of separation, we observed that most fractions had antiplatelet effects, which suggests that most peptides have some degree of antiplatelet effect. Following the inhibitory fractions, we purified and identified two new peptides, SSGE and DEE, by LC–electrospray ionization MS and peptide sequencing. Both peptides were highly hydrophilic. The concentrations to obtain 50% inhibition (IC50) of the aggregation intensity were approximately 480 and 460 μM, respectively, for SSGE and DEE. [Copyright &y& Elsevier]

Reports on a study in which gonadotropin (GTH) was purified from the pituitaries of the Pacific chinook salmon using a combination of stepwise ethanol precipitation and concanavalin-A affinity chromatography. Reverse-phase high-performance liquid chromatography; Screening of an enriched cDNA library for the β-GTH genes using two synthetic oligonucleotides; Effect of testosterone implantation on pituitary PTH and β-GTH mRNA; Corresponding increase in both the pituitary GTH and mRNA levels.

Tree nut along with peanut are among the most potent food allergens, responsible for frequently inducing the IgE-mediated hypersensitivity reaction. Our aim was identification, purification of Buchanania lanzan (Bl-11 kDa) protein along with characterization and assessment of allergenic potential of clinically relevant allergen. Further study was executed in clinical samples of sensitive patients, BALB/c mice, and in-vitro. A major IgE binding 11-kDa protein from Buchanania lanzan was purified by anion exchange chromatography, reverse phase high pressure liquid chromatography (RP-HPLC) and characterized using peptide mass fingerprinting (PMF). Buchanania lanzan (Bl-11 kDa) protein shows the pepsin resistance and depicts IgE interacting capacity to Buchanania lanzan allergic patient's sera as well as sensitized mice sera. It also showed increase in the allergic mediator's like IgE, IgG1, histamine levels in sensitized mice sera. Further study was carried out in-vitro (RBL-2H3 cells) and increased release mast cell degranulation mediators such as β-hexosaminidase, histamine, CysL and PGD2 in the culture supernatant was found. The activation of Th2 cytokines/transcription factors and expression of molecular markers in the downstream of mast cell signaling were up-regulated while the Th1 transcriptional factor (T-bet) was decreased in Bl-11 kDa protein treated mice. Conclusively, our study demonstrates Buchanania lanzan purified protein to be potential allergen that may generate an allergic reaction in sensitized individuals, and one of the most important IgE binding protein responsible for its allergenicity. Unlabelled Image • Bl mediated allergy is increasing due to its excessive usage in food preparations. • Bl-11 kDa protein shows the positive binding from Bl sensitized allergic patients. • Bl-11 kDa protein increased the levels of allergic parameters like IgE, IgG1, and Histamine etc. • Bl-11 kDa protein upregulated the Th2 mediated responses and activated the mast cell signaling resulting into degranulation. [ABSTRACT FROM AUTHOR]