Two-step Chromatography Research Articles

Purification and identification of antioxidative peptides from loach (Misgurnus anguillicaudatus) protein hydrolysate by consecutive chromatography and electrospray ionization-mass spectrometry

Loach protein was hydrolyzed by papain to obtain antioxidative peptides. The results showed that the loach protein hydrolysate (LPH) could scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) (IC 50 = 17.0 ± 0.54 mg/mL) and hydroxyl radicals (IC 50 = 2.64 ± 0.29 mg/mL). It could chelate cupric ion and inhibit the lipid peroxidation in a linoleic acid emulsion system. The hydrolysate was isolated and purified by ultrafiltration and consecutive chromatographic methods including ion-exchange chromatography, gel filtration chromatography and a two-step reverse high-performance liquid chromatography (RP-HPLC). The purified antioxidant peptide was identified as Pro-Ser-Tyr-Val (464.2 Da) using RP-HPLC connected on-line to an electrospray ionization (ESI) mass spectrometer. The purified peptide showed a 9.14-fold higher scavenging activity for hydroxyl radical compared with the crude LPH. Therefore, it is possible to produce natural antioxidative peptides from loach protein by enzymatic hydrolysis and purification.

Purification of antioxidative peptides prepared from enzymatic hydrolysates of tuna dark muscle by-product

Abstract To produce and identify bioactive peptides, two commercial enzymes, orientase (OR) and protease XXIII (PR) were used to hydrolyze tuna dark muscle by-product for up to 6 h, and the hydrolysates were evaluated for antioxidative properties. The results showed that, 60-min OR and 120-min PR hydrolysates possessed the highest antioxidative activity. Then, the protein hydrolysates were subjected to a Sephadex G-25 gel filtration chromatography, and the molecular weight of the peptide fractions which showed the highest antioxidative activity ranged from 390 to 1400 Da. The peptide fractions were further isolated using the two-step high-performance liquid chromatography (HPLC-1 and HPLC-2), and the amino acid sequences of the two antioxidative peptides from OR and PR hydrolysates were Leu–Pro–Thr–Ser–Glu–Ala–Ala–Lys–Tyr (978 Da) and Pro–Met–Asp–Tyr–Met–Val–Thr (756 Da), respectively. We thus conclude that antioxidative hydrolysates from tuna dark muscle by-product may be useful ingredients in food and nutraceutical applications.

High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus

BackgroundIn recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein) in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein). In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef.ResultsThe effect of three gene silencing viral suppressor proteins (P25 of Potato Virus X, P19 of either Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus) on Nef transient expression yield was evaluated. The P19 protein of Artichoke Mottled Crinckle virus (AMCV-P19) gave the highest expression yield in vacuum co-agroinfiltration experiments reaching 1.3% of total soluble protein, a level almost three times higher than that previously reported in stable transgenic plants. The high yield observed in the co-agroinfiltrated plants was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs) indicating an effective modulation of RNA silencing mechanisms by AMCV-P19. Interestingly, we also showed that expression levels in top leaves of vacuum co-agroinfiltrated plants were noticeably reduced compared to bottom leaves. Moreover, purification of Nef from agroinfiltrated tissue was achieved by a two-step immobilized metal ion affinity chromatography protocol with yields of 250 ng/g of fresh tissue.ConclusionWe demonstrated that expression level of HIV-1 Nef in plant can be improved using a transient expression system enhanced by the AMCV-P19 gene silencing suppressor protein. Moreover, plant-derived Nef was purified, with enhanced yield, exploiting a two-step purification protocol. These results represent a first step towards the development of a plant-derived HIV vaccine.

Antimicrobial Activity of Gallotannins Isolated from Mango (Mangifera indica L.) Kernels

Gallotannins were extracted from mango ( Mangifera indica L.) kernels with aqueous acetone (80%, v/v) and purified using liquid-liquid extraction and two-step low-pressure liquid chromatography (LPLC) on Sephadex LH-20. Analytical high-performance liquid chromatography and mass spectrometry confirmed the presence of hydrolyzable tannins with a degree of galloylation ranging from 4 to 9 and additionally revealed the presence of deca-, undeca-, and dodeca-O-galloylglucose. Further purification using two-step semipreparative HPLC resulted in three pure hydrolyzable tannins, penta-, hexa-, and hepta-O-galloylglucose, with antibacterial activity, as evidenced from the agar spot and critical dilution assays. Although the growth of lactic acid bacteria was not inhibited, the proliferation of Gram-positive food spoilage bacteria was prevented and the growth of Gram-negative Escherichia coli was reduced. Because bacterial growth could be restored by the addition of iron to the medium, this study strongly supports the view that the inhibitory effects of hydrolyzable tannins are due to their iron-complexing properties.

Characterization of Two Seryl-tRNA Synthetases in Albomycin-Producing Streptomyces sp. Strain ATCC 700974

The Trojan horse antibiotic albomycin, produced by Streptomyces sp. strain ATCC 700974, contains a thioribosyl nucleoside moiety linked to a hydroxamate siderophore through a serine residue. The seryl nucleoside structure (SB-217452) is a potent inhibitor of seryl-tRNA synthetase (SerRS) in the pathogenic bacterium Staphylococcus aureus, with a 50% inhibitory concentration (IC(50)) of approximately 8 nM. In the albomycin-producing Streptomyces sp., a bacterial SerRS homolog (Alb10) was found to be encoded in a biosynthetic gene cluster in addition to another serRS gene (serS1) at a different genetic locus. Alb10, named SerRS2 herein, is significantly divergent from SerRS1, which shows high homology to the housekeeping SerRS found in other Streptomyces species. We genetically and biochemically characterized the two genes and the proteins encoded. Both genes were able to complement a temperature-sensitive serS mutant of Escherichia coli and allowed growth at a nonpermissive temperature. serS2 was shown to confer albomycin resistance, with specific amino acid residues in the motif 2 signature sequences of SerRS2 playing key roles. SerRS1 and SerRS2 are comparably efficient in vitro, but the K(m) of serine for SerRS2 measured during tRNA aminoacylation is more than 20-fold higher than that for SerRS1. SB-217452 was also enzymatically generated and purified by two-step chromatography. Its IC(50) against SerRS1 was estimated to be 10-fold lower than that against SerRS2. In contrast, both SerRSs displayed comparable inhibition kinetics for serine hydroxamate, indicating that SerRS2 was specifically resistant to SB-217452. These data suggest that mining Streptomyces genomes for duplicated aminoacyl-tRNA synthetase genes could provide a novel approach for the identification of natural products targeting aminoacyl-tRNA synthetases.

The alpha 2 chain of collagen type VI sequesters latent proforms of matrix-metalloproteinases and modulates their activation and activity

The extracellular matrix (ECM) attracts increasing attention as a store of biologically active molecules and as a reservoir of potent cell signalling molecules released by proteolytic action. Both, cytokines and proteases mediating such release are sequestered in the ECM. Here, we found matrix metalloproteinase (MMP) proforms closely associated with collagenous septae in fibrotic liver tissue, and we screened immobilized human placenta-derived collagen chains and other ECM proteins for MMP-binding activity. Following the establishment of a novel highly-efficient two-step chromatography procedure for the isolation of the purified α-chains of the pepsin-resistant triple-helical CVI fragment (CVI/PR) solid phase and surface plasmon resonance binding studies were performed. We identified the triple-helical domain of the α2 chain of microfilamentous CVI α2(VI) as having nanomolar affinity for the collagenases proMMP-1, -8 , -13 and stromelysin-1 (MMP-3), thus extending the repertoire of pericellular and substrate-based interactions of MMPs. Enzymatic activity assays enabled the correlation of MMP activity with CVI binding, in that α2(VI) chain-mediated inhibition of enzymatic activity is accompanied by increased binding. Similar results were shown for the gelatinase proMMP-9, whereas for proMMP-2, the α2(VI) chain at low concentrations seems to interfere with prodomain binding resulting in enhanced activity without scission of the prodomain. Stable complexes of proMMP-2 and α2(VI) chain competed with gelatinase binding to the preferred ligand, collagen type I. In conclusion, the α2(VI) chain modulates MMP availability by sequestering proMMPs in the ECM, blocking proteolytic activity. Therefore, CVI and especially its α2(VI) chain might serve as a lead structure for MMP-based therapeutics which modulates the action of these matrix components, e.g. in fibrosis and cancer.

Purification of adenoviral vectors by combined anion exchange and gel filtration chromatography

Adenoviral vectors are used extensively in human gene therapy trials and in vaccine development. Large-scale GMP production requires a downstream purification process, and liquid chromatography is emerging as the most powerful mode of purification, enabling the production of vectors at a clinically relevant scale and quality. The present study describes the development of a two-step high-performance liquid chromatography (HPLC) process combining anion exchange (AIEX) and gel filtration (GF) in comparison with the caesium chloride density gradient method. HEK-293 cells were cultured in ten-layer CellStacks() and infected with 10 pfu/cell of adenoviral vector expressing green fluorescent protein (Ad5-GFP). Cell-bound virus was harvested and benzonase added to digest DNA, crude lysate was clarified by centrifugation and filtration prior to HPLC. Chromatography fractions were added to HEK-293 cells and GFP expression measured using a fluorescent plate reader. Using AIEX then GF resulted in an adenoviral vector with purity comparable to Ad5-GFP purified by CsCl, whereas the reverse process (GF-AIEX) showed a reduced purity by electrophoresis and required further buffer exchange of the product. The optimal process (AIEX-GF) resulted in a vector yield of 2.3 x 10(7) pfu/cm(2) of cell culture harvested compared to 3.3 x 10(7) pfu/cm(2) for CsCl. The process recovery for the HPLC process was 36% compared to 27.5% for CsCl and total virion to infectious particle ratios of 18 and 11, respectively, were measured. We present a simple two-step chromatography process that is capable of producing high-quality adenovirus at a titre suitable for scale-up and clinical translation.

Purification, crystal structure and antimicrobial activity of phenazine-1-carboxamide produced by a growth-promoting biocontrol bacterium,Pseudomonas aeruginosaMML2212

To purify and characterize an antimicrobial compound produced by a biocontrol bacterium, Pseudomonas aeruginosa MML2212, and evaluate its activity against rice pathogens, Rhizoctonia solani and Xanthomonas oryzae pv. oryzae. Pseudomonas aeruginosa strain MML2212 isolated from the rice rhizosphere with wide-spectrum antimicrobial activity was cultured in Kings'B broth using a fermentor for 36 h. The extracellular metabolites were isolated from the fermented broth using ethyl acetate extraction and purified by two-step silica-gel column chromatography. Three fractions were separated, of which a major compound was obtained in pure state as yellow needles. It was crystallized after dissolving with chloroform followed by slow evaporation. It is odourless with a melting point of 220-222 degrees C. It was soluble in most of the organic solvents and poorly soluble in water. The molecular mass of purified compound was estimated as 223.3 by mass spectral analysis. Further, it was characterized by IR, (1)H and (13)C NMR spectral analyses. The crystal structure of the compound was elucidated for the first time by X-ray diffraction study and deposited in the Cambridge Crystallographic Data Centre (http://www.ccde.com.ac.uk) with the accession no. CCDC 617344. The crystal compound was undoubtedly identified as phenazine-1-carboxamide (PCN) with the empirical formula of C(13)H(9)N(3)O. As this is the first report on the crystal structure of PCN, it provides additional information to the structural chemistry. Furthermore, the present study reports the antimicrobial activity of purified PCN on major rice pathogens, R. solani and X. oryzae pv. oryzae. Therefore, the PCN can be developed as an ideal agrochemical candidate for the control of both sheath blight and bacterial leaf blight diseases of rice.

Coexpression of ω subunit in E. coli is required for the maintenance of enzymatic activity of Xanthomonas campestris pv. campestris RNA polymerase

In this study, plasmid pBBad22K was modulated to be able to coexpress the subunits of Xanthomonas campestris pv. campestris ( Xcc) core RNA polymerase (RNAP) in an Escherichia coli host. The subunit-encoding genes of Xcc core RNAP were PCR-amplified respectively to convert into gene cassettes in which an intact subunit-encoding gene and a ribosome binding site (RBS) preceding the gene were contained, and were then cloned one by one into pBBad22K. In addition, a hexahistidine tag (His-tag) was introduced into the C-terminus of α subunit-encoded rpoA during PCR for facilitating purification of Xcc core RNAP. The resultant vectors, pBC-CBA and pBC-CBAZ, were used for overproduction of Xcc core RNAP lacking or containing ω, respectively. The assembly of Xcc core RNAP subunits that were coexpressed from these vectors was demonstrated after purification via two-step column chromatography. The yield of Xcc core RNAP containing ω had a 67% increase compared with that of one lacking ω, indicating that ω promotes the assembly of Xcc core RNAP. In addition, Xcc core RNAP lacking ω showed a 13-fold decrease in enzymatic activity in comparison with that containing ω. Promoter-specific transcription assays by recombinant Xcc core RNAP reconstituted with external added σ factor showed that the absence of ω debilitates the transcriptional activity of Xcc RNAP. Our results demonstrated that ω is not only capable of strengthening the stability, but is also required for the maintenance of enzymatic activity of Xcc RNAP.

Masking effect of anti-androgens on androgenic activity in European river sediment unveiled by effect-directed analysis

This study shows that the androgen receptor agonistic potency is clearly concealed by the effects of androgen receptor antagonists in a total sediment extract, demonstrating that toxicity screening of total extracts is not enough to evaluate the full in vitro endocrine disrupting potential of a complex chemical mixture, as encountered in the environment. The anti-androgenic compounds were masking the activity of androgenic compounds in the extract with relatively high anti-androgenic potency, equivalent to 200 nmol flutamide equivalents/g dry weight. A two-step serial liquid chromatography fractionation of the extract successfully separated anti-androgenic compounds from androgenic compounds, resulting in a total androgenic potency of 3,820 pmol dihydrotestosterone equivalents/g dry weight. The fractionation simplified the chemical identification analysis of the original complex sample matrix. Seventeen chemical structures were tentatively identified. Polyaromatic hydrocarbons, a technical mixture of nonylphenol and dibutyl phthalate were identified to contribute to the anti-androgenic potency observed in the river sediment sample. With the GC/MS screening method applied here, no compounds with AR agonistic disrupting potencies could be identified. Seventy-one unidentified peaks, which represent potentially new endocrine disrupters, have been added to a database for future investigation.

715. A Rapid, Scalable, Two-Step Column Chromatography Purification of rAAV Vectors

715. A Rapid, Scalable, Two-Step Column Chromatography Purification of rAAV Vectors

Recognition ofCandida albicansAls3 by the germ tube-specific monoclonal antibody 3D9.3

Monoclonal antibody 3D9.3 (MAb 3D9.3) reacts with the surface of Candida albicans germ tubes and recognizes a protein epitope. We used a two-step chromatography procedure to purify and identify the antigen (3D9) from C. albicans strain 66396 germ tubes. MAb 3D9.3 recognized two intense protein bands at 140 and 180 kDa. A comparative analysis between theoretical and experimental mass spectrum peaks showed that both bands corresponded to Als3. This conclusion was supported by lack of reactivity between MAb 3D9.3 and an als3Delta/als3Delta mutant strain, and the fact that an immunoglobulin preparation enriched for Als3 specificity recognized the purified 3D9 antigen. PCR demonstrated that C. albicans strain 66396 has two different-sized ALS3 alleles that correspond to the two purified protein bands. Strain- and species-specificity of the 3D9 epitope were studied with various C. albicans strains and Candida species, such as closely related Candida dubliniensis. The 3D9 epitope was detected only in C. albicans, demonstrating the utility of MAb 3D9.3 for differentiation between C. albicans and C. dubliniensis. Adhesion assays demonstrated that MAb 3D9.3 blocks adhesion of C. albicans germ tubes to human buccal epithelial cells and vascular endothelial cells.

Antioxidative properties of peptides prepared from tuna cooking juice hydrolysates with orientase (Bacillus subtilis)

Abstract The antioxidative activity of isolated peptides from of tuna cooking juice hydrolysates by orientase was determined. The results showed that, among the hydrolysates obtained under different hydrolysis time, the highest antioxidative activity and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging capacity were presented at 60 min. Then, the protein hydrolysate was subjected to a Sephadex G-25 gel filtration chromatography, and five major fractions were obtained. Peptide fractions ranging from 400 to 1500 Da showed the highest antioxidative activity among all fractions. The peptide fractions were further isolated using the two-step high performance liquid chromatography (HPLC-1 and HPLC-2), and six and three fractions were obtained, respectively. The final three antioxidative peptides comprised 4–10 amino acids sequences were observed, and the structures of the peptides were Pro-Val-Ser-His-Asp-His-Ala-Pro-Glu-Tyr (1305 Da), Pro-Ser-Asp-His-Asp-His-Glu (938 Da), and Val-His-Asp-Tyr (584 Da), respectively. We thus conclude that antioxidative hydrolysates from tuna cooking juice may be useful ingredients in food and nutraceutical applications.

Phytochemistry of cimicifugic acids and associated bases in Cimicifuga racemosa root extracts

Earlier studies reported serotonergic activity for cimicifugic acids (CA) isolated from Cimicifuga racemosa. The discovery of strongly basic alkaloids, cimipronidines, from the active extract partition and evaluation of previously employed work-up procedures has led to the hypothesis of strong acid/base association in the extract. Re-isolation of the CAs was desired to permit further detailed studies. Based on the acid/base association hypothesis, a new separation scheme of the active partition was required, which separates acids from associated bases. A new 5-HT(7) bioassay guided work-up procedure was developed that concentrates activity into one partition. The latter was subjected to a new two-step centrifugal partitioning chromatography (CPC) method, which applies pH zone refinement gradient (pHZR CPC) to dissociate the acid/base complexes. The resulting CA fraction was subjected to a second CPC step. Fractions and compounds were monitored by (1)H NMR using a structure-based spin-pattern analysis facilitating dereplication of the known acids. Bioassay results were obtained for the pHZR CPC fractions and for purified CAs. A new CA was characterised. While none of the pure CAs was active, the serotonergic activity was concentrated in a single pHZR CPC fraction, which was subsequently shown to contain low levels of the potent 5-HT(7) ligand, N(omega)-methylserotonin. This study shows that CAs are not responsible for serotonergic activity in black cohosh. New phytochemical methodology (pHZR CPC) and a sensitive dereplication method (LC-MS) led to the identification of N(omega)-methylserotonin as serotonergic active principle.

Ecotoxicity of the degradation products of triphenylborane pyridine (TPBP) antifouling agent

Triphenylborane pyridine (TPBP) is an alternative to organotin antifouling compounds. This work aimed to identify the unknown Peak #1, and to evaluate the ecotoxicity of TPBP and its degradation products. Peak #1 was produced from TPBP dissolved in acetonitrile under UV-A photolysis using a high-pressure mercury lamp. The Peak #1 fraction was purified using two-step column chromatography from a TPBP–acetonitrile solution. The major compound of the fraction was identified as being biphenyl from the 1H NMR and 13C NMR spectra. The ecotoxicity of four degradation products (diphenylborane hydroxide, phenylborane dihydroxide, phenol, and biphenyl) and TPBP towards two marine planktons were assessed. The 48 h LC 50 values of the crustacean, Artemia salina, were 0.13 mg L −1 for TPBP, 14 mg L −1 for biphenyl, 17 mg L −1 for phenol, and >50 mg L −1 for the other degradation products. The 72 h EC 50 values of the diatom, Skeletonema costatum, were 0.0022 mg L −1 for TPBP, 1.2 mg L −1 for biphenyl, and >2 mg L −1 for the other degradation products. Thus, the ecotoxicity of biphenyl and the other degradation products were not high compared to the parent compound, TPBP.

Purification, characterization and cloning of a ricin B-like lectin from mushroom Clitocybe nebularis with antiproliferative activity against human leukemic T cells

Background Lectins are a diverse group of carbohydrate-binding proteins exhibiting numerous biological activities and functions. Methods Two-step serial carbohydrate affinity chromatography was used to isolate a lectin from the edible mushroom clouded agaric ( Clitocybe nebularis). It was characterized biochemically, its gene and cDNA cloned and the deduced amino acid sequence analyzed. Its activity was tested by hemagglutination assay and carbohydrate-binding specificity determined by glycan microarray analysis. Its effect on proliferation of several human cell lines was determined by MTS assay. Results A homodimeric lectin with 15.9-kDa subunits agglutinates human group A, followed by B, O, and bovine erythrocytes. Hemagglutination was inhibited by glycoprotein asialofetuin and lactose. Glycan microarray analysis revealed that the lectin recognizes human blood group A determinant GalNAcα1–3(Fucα1–2)Galβ-containing carbohydrates, and GalNAcβ1–4GlcNAc ( N, N'-diacetyllactosediamine). The lectin exerts antiproliferative activity specific to human leukemic T cells. Conclusions The protein belongs to the ricin B-like lectin superfamily, and has been designated as C. nebularis lectin (CNL). Its antiproliferative effect appears to be elicited by binding to carbohydrate receptors on human leukemic T cells. General significance CNL is one of the few mushroom ricin B-like lectins that have been identified and the only one so far shown to possess immunomodulatory properties.

Improved purification of recombinant adenoviral vector by metal affinity membrane chromatography

The increasing importance of adenoviral vectors for gene therapy clinical trials necessitates the development of processes suitable for large-scale and commercial production of adenovirus. Here, we evaluated a novel purification process combining an anion-exchange chromatography and an immobilized metal affinity membrane chromatography for the purification of recombinant adenovirus. Adenovirus was initially purified from clarified infectious lysate by anion-exchange chromatography using Q Sepharose XL resin and further polished using a Sartobind IDA membrane unit charged with Zn 2+ ions as affinity ligands. The metal affinity membrane chromatography efficiently removed residual host cell impurities that co-eluted with adenovirus during the previous anion-exchange chromatography step. The metal affinity membrane chromatography also separated defective adenovirus particles from the infectious adenovirus fraction. Furthermore, the metal affinity membrane chromatography showed an improved yield, when compared with a conventional bead-based metal affinity chromatography. The purity and specific activity of the adenovirus prepared using this two-step chromatography was comparable to those of adenovirus produced by the conventional CsCl density centrifugation. Therefore, our data provide an improved method for the purification of adenoviral vectors for clinical applications.

Gene Cloning and Heterologous Expression of a Novel Endoglucanase, Swollenin, fromTrichoderma pseudokoningiiS38

The coding sequence of a novel cellulolytic factor, swollenin, was isolated from the cellulolytic fungus Trichoderma pseudokoningii S38. The full-length swo2 gene encodes a protein of 494 amino acids with a calculated molecular mass of 51,393 Da, which includes a putative 22-amino-acid signal peptide. Sequence analysis revealed significant identity between isolated swollenin and that from Trichoderma reesei. The swollenin gene was further expressed and purified in T. reesei QM9414. The expressed swollenin protein was consequently purified by two-step ion exchange chromatography. The purified swollenin had subtle hydrolytic activities on xylan and yeast cell wall glucan, while no apparent activities on carboxymethy cellulose, cotton fiber, filter paper, or cellulose powder CF11 were observed. These results indicate that although swollenin maintains unidentified glycohydrolytic activities, it is inactive against beta-1,4-glycosidic bonds in cellulose. Its exact role in lignocellulose hydrolysis calls for further analysis.

Structural classification of acyl‐substituted Quillaja saponins by electrospray ionisation ion trap multiple‐stage mass spectrometry in combination with multivariate analysis

Thirty-eight saponins in two chromatographic fractions (QH-B and QH-C) from Quillaja saponaria Molina have been separated by a two-step high-performance liquid chromatography (HPLC) procedure and investigated by electrospray ionisation ion trap multiple-stage mass spectrometry (ESI-ITMS(n)) in positive ion mode. MS(2) and MS(3) spectra of the compounds were investigated by principal component analysis (PCA) and could be classified by partial least squares - discriminant analysis (PLS-DA) according to the structures of the oligosaccharides at C-3 and C-28 of the saponins. Four minor components with novel structures were found in a previously non-investigated fraction of QH-C. The structures of two of these components, J1 and J1a, were predicted by PLS-DA whereas the structures of the two others, J2 and J3, were only partly predicted. The structures of J1 and J1a were composed of structural elements found in the 34 known saponins whereas a new acyl substituent, not included in the training set used for calibration of the PLS-DA models, was found in J2 and J3, making these two components outliers. The complete structures of the four components were confirmed by monosaccharide analysis, MS(n) data and (1)H NMR spectroscopy.

Colombistatin: a disintegrin isolated from the venom of the South American snake (Bothrops colombiensis) that effectively inhibits platelet aggregation and SK-Mel-28 cell adhesion

Snake venoms are complex mixtures of proteins, which affect the vital biologic systems of prey, as well as humans. Envenomation leads to immobilization by paralysis, cardiac, and circulatory failure. These same venom proteins that cause havoc in the physiologic system could be used as therapeutic agents. Disintegrins and disintegrin-like proteins are molecules found in the venom of four snake families (Atractaspididae, Elapidae, Viperidae, and Colubridae). The disintegrins are non-enzymatic proteins that inhibit cell-cell interactions, cell-matrix interactions, and signal transduction. These proteins may have potential in the treatment of strokes, heart attacks, cancers, osteoporosis, and diabetes. The present study describes the isolation and characterization of a disintegrin (colombistatin) found in the venom of the Venezuelan snake mapanare (Bothrops colombiensis). Colombistatin was purified by a two-step high-performance liquid chromatography procedure, which included reverse phase C18 and size exclusion protein Pak 60. Colombistatin inhibited ADP-induced platelet aggregation, human urinary (T24) and skin melanoma (SK-Mel-28) cancer cell adhesion to fibronectin, and cell migration. Colombistatin contained 72 amino acids with a mass of 7.778 kDa as determined by mass spectrometry. Colombistatin could be used as a therapeutic tool in the treatment of melanoma cancers and also thrombotic diseases.