CM-cellulose Ion-exchange Chromatography Research Articles

Cytotoxic Activity of NN-32 Toxin from Indian Spectacled Cobra Venom on Human Breast Cancer Cell Line

Animal venoms and toxins are potential bioresources that have been known to mankind as a therapeutic tool for more than a century through folk and traditional medicine. The present study was an effort to establish the anticancer activity of the purified protein toxin (NN-32) from Indian Spectacled Cobra (Naja naja) venom in human breast cancer cell line. Isolation and purification of NN-32 was done through CM-cellulose ion exchange chromatography and RP- HPLC. Molecular weight was found out by SDS-PAGE. The anti-leukemic activity using MCF-7 cell line was established through cytotoxicity study. NN-32 was eluted with 0.5M NaCl on CM-cellulose ion exchange chromatography. SDS-PAGE molecular weight was found to be 6.7 KDa. NN-32 produced time and dose dependent cell (MCF-7) growth inhibition. It exhibited DNA fragmentation and comet formation in MCF-7 cells. NN-32 produced membrane disruption, blebbing and nuclear disintegration in MCF-7 cells observed through scanning electron microscopy. NN-32 produced apoptosis, cell cycle arrest at G1 phase. NN-32 induced apoptosis in leukemic cells was followed through caspase 3 and 9 pathway activation. It may be concluded that NN-32, a 6.7 KDa protein purified from Naja naja venom would be a novel pro-apoptotic agent that induced cancer cell killing through p53 and caspase pathway. It is expected that this study may add new information on anticancer effects of Naja naja snake venom, which may be utilized for future drug development clue against cancer.

Anti-osteoarthritic activity of Bungarus fasciatus venom fraction BF-F47 involving molecular markers in the rats

A heat stable protein BF-F47 was purified from the crude venom of Bungarus fasciatus by CM cellulose ion exchange chromatography and HPLC. Osteoarthritis (OA) was developed in male albino Wistar rats by collagenase injection. BF-F47 treatment significantly restored urinary hydroxyproline and glucosamine in OA rats. Serum acid phosphatase, alkaline phosphatase, creatinine and serum molecular markers TNF-α, IL-1β, IL-17, cytokine induced neutrophil chemoattractant-1, matrix metalloproteinase-1, cathepsin-K, osteocalcin and PGE2 were also significantly altered. BF-F47 showed partial restoration of osteoarthritis joints. Thus, BF-F47 induced anti-osteoarthritic activity in Wistar rats acted through molecular markers of arthritis and inflammation.

Russell’s Viper Venom Purified Toxin Drct-II Inhibits the Cell Proliferation and Induces G1 Cell Cycle Arrest in Human Leukemic Cancer Cells

The present study was an effort to establish the anticancer activity of the purified protein toxin (drCT-II) from Indian Russell’s viper (Daboia russelli russelli) venom in leukemic cell line and animal model. Isolation and purification of drCT-II was done through CM-cellulose ion exchange chromatography and RP- HPLC. SDS- PAGE molecular weight and first 20 amino acid sequence of drCT-II was done. The anti-leukemic activity using U937 and K562 cell line was established through cytotoxicity, apoptosis, cell cycle study, morphology, cancer marker proteins. The mean survival time of EAC induced male albino mice was established. Human lymphocyte cytotoxicity was done. drCT-II was eluted with 0.1 M NaCl on CM-cellulose ion exchange chromatography. On RP-HPLC, drCT-II produced single peak with retention time of 14.6 min. SDS-PAGE molecular weight was found to be 6.6 KDa and the first 20 amino acid sequence was found to be LQXNKLVPIASKTXPPGKNL. drCT-II produced time and dose dependent cell (U937 and K562) growth inhibition. The IC50 was found to be 35.5 μg/ml for U937 cell and 48.2 μg/ml for K562 cells. drCT-II produced membrane disruption, blebbing and nuclear disintegration in U937 and K562 cells observed through confocal and scanning electron microscopy. It exhibited DNA fragmentation and comet formation in leukemic cells. drCT-II produced apoptosis, cell cycle arrest at G1 phase and increased the expression of P21, P27 and P53. drCT-II induced apoptosis in leukemic cells was followed through caspase 3 and 9 pathway activation. EAC cell growth in male albino mice was significantly inhibited by drCT-II, thus increased the mean survival time. drCT-II significantly reduced the human lymphocyte count (in culture). It may be concluded that drCT-II, a 6.6 KDa protein purified from Daboia russelli russelli venom would be a novel pro-apoptotic agent that induced cancer cell killing through p53 and caspase pathway.

A cytotoxic protein (BF-CT1) purified from Bungarus fasciatus venom acts through apoptosis, modulation of PI3K/AKT, MAPKinase pathway and cell cycle regulation

BF-CT1, a 13 kDa protein isolated from Bungarus fasciatus snake venom through CM cellulose ion exchange chromatography at 0.02 M NaCl salt gradient showed cytotoxicity in in vitro and in vivo experimental models. In in vivo Ehrlich ascites carcinoma (EAC) induced BALB/c mice model, BF-CT1 treatment reduced EAC cell count significantly through apoptotic cell death pathway as evidenced by FACS analysis, increased caspase 3, 9 activity and altered pro, antiapoptotic protein expression. BF-CT1 treatment caused cell shrinkage, chromatin condensation and induced apoptosis through increased caspase 3, caspase 9 activity, PARP cleavage and down regulation of heat shock proteins in U937 leukemic cell line. Cytosolic cytochrome C production was increased after BF-CT1 treatment upon U937 cell line. BF-CT1 treated U937 cell showed cell cycle arrest at sub G1 phase through cyclin D and CDK down regulation with up regulation of p15 and p16. It also down regulated PI3K/AKT pathway and MAPkinase pathway and promoted apoptosis and regulated cell proliferation in U937 cells. BF-CT1 prevented angiogenesis in in vitro U937 cell line through decreased VEGF and TGF-β1 production.

Purification and characterization of a subtilisin-like proteinases secreted in the stationary growth phase of Bacillus amyloliquefaciens H2

Proteinases secreted during the early and late stationary phases have been isolated from the culture liquid of Bacillus amyloliquefaciens H2 using CM-cellulose ion-exchange chromatography with subsequent FPLC on a Mono S column. Considering the character of hydrolysis of specific chromogenic substrates and the type of inhibition, these enzymes were identified as subtilisin-like proteinases. The molecular weight of both proteinases is 29 kD. The proteolytic activity of the proteinases secreted during the early and late stationary phases towards the synthetic substrate Z-Ala-Ala-Leu-pNA was maximal at pH 8.5 and 9.0, respectively. The maximal activity of both proteinases was observed at 37 degrees C, and the proteins were stable within the pH range of 7.2-9.5. The subtilisin-like proteinases from B. amyloliquefaciens were shown to catalyze synthesis of peptide bonds.

Studies on a thermostable alpha-amylase from the thermophilic fungus Scytalidium thermophilum.

An alpha-amylase produced by Scytalidium thermophilum was purified using DEAE-cellulose and CM-cellulose ion exchange chromatography and Sepharose 6B gel filtration. The purified protein migrated as a single band in 6% PAGE and 7% SDS-PAGE. The estimated molecular mass was 36 kDa (SDS-PAGE) and 49 kDa (Sepharose 6B). Optima of pH and temperature were 6.0 and 60 degrees C, respectively. In the absence of substrate the purified alpha-amylase was stable for 1 h at 50 degrees C and had a half-life of 12 min at 60 degrees C, but was fully stable in the presence of starch. The enzyme was not activated by several metal ions tested, including Ca(2+) (up to 10 mM), but HgCl(2 )and CuCl(2) inhibited its activity. The alpha-amylase produced by S. thermophilum preferentially hydrolyzed starch, and to a lesser extent amylopectin, maltose, amylose and glycogen in that order. The products of starch hydrolysis (up to 6 h of reaction) analyzed by thin layer chromatography, showed oligosaccharides such as maltotrioses, maltotetraoses and maltopentaoses. Maltose and traces of glucose were formed only after 3 h of reaction. These results confirm the character of the enzyme studied to be an alpha-amylase (1,4-alpha-glucan glucanohydrolase).

Isolation and Partial Characterization of Lectin from the Venom of Vietnamese Scorpion Buthus occitanussp.

A lectin was isolated from the venom of scorpion Buthus occitanussp. by means of Sephadex G-50 gel filtration and CM-cellulose ion exchange chromatography. The homogeneous lectin preparation consisted of homodimeric molecules with a subunit Mrof 9.3 kDa. Glycine, alanine, and serine dominated in the lectin amino acid composition. The lectin was a glycoprotein containing 20% carbohydrates (predominantly mannose and glucose). Trypsin-treated murine erythrocytes agglutinated at a lectin concentration of 32 μg/ml. Hemagglutination was inhibited by carbohydrates (L-fucose > D-glucose > L-rhamnose > D-xylose). The lectin revealed no phospholipase or hyaluronidase, nor toxic activity.

Purification and Characterisation of Limit Dextrinase Inhibitors from Barley

Abstract Two inhibitors of malt limit dextrinase were purified from a crude barley extract (cv. Harrington) by CM cellulose ion exchange chromatography and chromatofocusing. The inhibitors were heat-stable proteins of Mr approximately 15k and isoelectric points of 6·7 (low pI inhibitor) and 7·2 (high pI inhibitor). Both inhibitors were active over a wide pH range, and were most effective at pH 5·5 to 6·5, the pH optimum of the limit dextrinase enzyme. Inactivation of the limit dextrinase enzyme by either inhibitor could be reversed by warning the complex at 40°C in the presence of reducing agents.

PURIFICATION AND CHARACTERIZATION OF β-GALACTOSIDASE OF `FUJI' APPLES

β-Galactosidase was purified from apple, Malus domestica Borkh, cv. Fuji by gel filtration, CM cellulose ion exchange chromatography, and characterizied by means of several biochemical methods. One form of β-Galactosidase was detected and the Km and Vmax values were calculated to be 0.035 and 0.036 mM with para-nitrophenyl-β-D-galactoside lmM/15min., respectively. The β-galactosidase was active between pH 3 and 7 with the optimum pH of about 4-5. The stable temperature for β-galactosidase was lower than 45°C with 30°C optimum. The β-galactosidase activities were inhibited by Ag*. EDTA and SDS.

Multiple forms of endopeptidase activity from Jojoba seeds

The cotyledons of 27 day post-germination jojoba seedlings ( Simmondsia chinensis) contained five distinct endopeptidase activities separable by DEAE Bio-Gel and CM-cellulose ion exchange chromatography. The endopeptidases were purified 108- to 266-fold and their individuality was confirmed by activity-specific assays in native acrylamide gels along with differences in their M r and catalytic properties. The five endopeptidases, which showed activity on model substrates and protein, were named EP Ia, EP Ib, EP II, EP III and EP IV. EP Ia was a serine proteinase with a pH optimum of ca 8 and M r of 58 000. EP Ib, II and III were discrete cysteine proteinases showing pH optima of ca 6.8, 6.0 and 5.4 and M r of 41 000, 47 000 and 35 000 respectively. EP IV was an aspartic acid proteinase with a ca pH optimum of 3.5 and M r of 33 000.

Isolation and characterization of fatty acid binding proteins from mammary tissue of lactating rats.

A protein fraction with fatty acid binding activity has been isolated from mammary tissue from lactating rats by a process involving DEAE-cellulose ion-exchange chromatography, heat treatment, CM-cellulose ion-exchange chromatography and finally ammonium sulphate precipitation. The purified fraction migrated as a single band on SDS/polyacrylamide-gel electrophoresis with an apparent molecular mass of 14400. However, when this protein fraction was electrophoresed under non-dissociating conditions, two species were observed in a 4:1 ratio. The two components were separated using h.p.l.c. Both bind fatty acids and appear to have similar amino acid compositions although exhibiting different pI values of 4.8 and 4.9. The mammary fatty acid binding proteins appear to be very similar to the fatty acid binding protein isolated from rat heart based on the electrophoretic mobilities and amino acid composition. The major mammary form (pI 4.9) has been partially sequenced and the amino acid sequences obtained can be aligned with 67 residues of the revised rat heart amino acid sequence [Heuckeroth, Birkenmeier, Levin & Gordon (1987) J. Biol. Chem. 262, 9709-9717]. Both mammary species also showed immunochemical identity to rat heart fatty acid binding protein when tested with an anti-serum raised against the heart protein. Anti-sera raised against the minor mammary form (pI 4.8) specifically precipitated this form under non-denaturing conditions but both forms after they had been denatured. Quantitative immunoassays using the anti-(heart fatty acid binding protein) serum showed that concentrations of the fatty acid binding proteins present in mammary cytosols increase during lactation and increase further after feeding a high-fat diet.

Isolation, characterization, and amino-terminal amino acid sequence analysis of human neutrophil cathepsin G from normal donors

Human neutrophil cathepsin G from normal donors has been purified 82-fold using an isolation procedure which included sequential sodium chloride extraction, Aprotonin-Sepharose affinity chromatography, CM-cellulose ion-exchange chromatography, and AcA 44 gel filtration chromatography. The inclusion of this last purification step was crucial for separating inactive lower molecular weight species from the active forms of neutrophil cathepsin G and resulted in a higher specific activity of the final preparation. SDS polyacrylamide gradient gel electrophoresis of the purified reduced protein demonstrated three discrete polypeptides of M r 31,000, 30,000, and 29,500. Peptide analysis of tryptic digests indicated that these three polypeptides are structurally related to each other and represent microheterogeneity of the purified protein. The cathepsin G peptide maps were distinctly different from the peptide maps of neutrophil elastase. The apparent isoelectric points of these forms as determined by two-dimensional electrophoresis was approximately 8.0. Utilizing microsequencing techniques, the first 25 residues of normal neutrophil cathepsin G have been determined and shown to be identical (except for residue 11) with the sequence of 21 residues of cathepsin G isolated from leukemic myeloid cells. A high degree of homology was found when the amino-terminal regions of neutrophil cathepsin G, rat mast cell protease II (65%) and two human serine proteinases, factor D (52%) and neutrophil elastase (48%), were compared. A precipitating monospecific antiserum to cathepsin G was produced by repeated immunizations of guinea pigs. This antiserum has been used in immunoblotting experiments to demonstrate that the intracellular form(s) of this enzyme is the same approximate M r as the purified enzyme, and to develop a solid-phase radioimmunoassay for measuring neutrophil cathepsin G in the range 5–50 ng/ml.

Isolation, characterization, and amino-terminal amino acid sequence analysis of human neutrophil elastase from normal donors

Human neutrophil elastase from normal donors has been purified using an isolation procedure which included sequential sodium chloride extraction, Aprotinin-Sepharose affinity chromatography, CM-cellulose ion-exchange chromatography, and AcA 44 gel filtration chromatography. The inclusion of this last purification step was crucial for separating inactive lower molecular weight species from the active forms of neutrophil elastase and resulted in a higher specific activity of the final preparation. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis of the reduced purified protein demonstrated three polypeptides of M r 31,000, 28,000, and 27,500. Four polypeptides were resolved on acid gel electrophoresis; each of the four possessed amidolytic activity. Furthermore, peptide analysis of Staphylococcus aureus V8 protease digests indicated that these polypeptides are structurally related to aach other and represent microheterogeneity of the purified protein. The apparent isoelectric points of these four forms as determined by two-dimensional electrophoresis range from 6.1 to 6.7. By utilizing microsequencing techniques, the first 40 residues of neutrophil elastase have been determined and compared with the reported sequence of elastase isolated from leukemic myeloid cells. In addition, a high degree of homology was found within the amino-terminal regions of neutrophil elastase and the serine proteinases porcine elastase, bovine chymotrypsin, human factor D, and the β chain of plasmin.

Mouse bone collagenase inhibitor: Purification and partial characterization of the inhibitor from mouse calvaria cultures

The organ culture of neonatal mouse calvaria produced both collagenase and collagenase inhibitor. The inhibitor was purified by a series of column chromatographies: DEAE-cellulose and CM-cellulose ion-exchange chromatography, concanavalin A-Sepharose and heparin-Sepharose affinity chromatography, and finally by Sephacryl S-200 gel filtration. The purified inhibitor migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a molecular mass of 28,000. The inhibitor was purified 140-fold to a specific activity of 163 units/mg with a yield of 18% over the first step of the purification by DEAE-cellulose chromatography. The inhibitor stained positively for carbohydrate with periodic acid-Schiff's reagent indicating, in conjunction with its affinity to concanavalin A, that the inhibitor is a glycoprotein. In addition to mouse bone collagenase, this inhibitor also inhibited chick bone, rat bone, rabbit corneal, and human gingival collagenase, but did not inhibit bacterial collagenase.

Studies on histone acetyltransferase. Partial purification and basic properties.

A rapid and reproducible method for the purification of rat liver histone acetyltransferase is presented. Extraction of nuclei in low salt, followed by phenyl-Sepharose hydrophobic affinity chromatography, G-200 gel filtration in the presence of 1 M urea, CM-cellulose ion exchange and acetyllysine affinity chromatography minimize exposure of the enzyme to high salt. Evidence is provided which indicates that the instability of the enzyme activity is due in part to hydrophobic interactions. The molecular weight of the enzyme is 96,000 as judged by gel filtration. In agreement with others, the enzyme is unstable in the presence of divalent cations, although a requirement for low concentration of Mg2+ or Ca2+ was observed. The enzyme is also sensitive to sulfhydryl blocking agents and is susceptible to rapid thermal denaturation at 37 and 45 degrees C (t1/2 = 22.2 and 9.54 min, respectively). The optimum pH and the energy of activation for the reaction were pH 7.5 and 5230 +/- 378 cal/mol, respectively. In the presence of all five histones, the enzyme catalyzes the acetylation in the order of H3 greater than H4 greater than H2b greater than H2a greater than H1 and appears to operate in a nonprocessive manner. While no other isozymic forms of nuclear acetyltransferase were detected, the enzyme exhibits the properties of both nuclear isozymic forms which have been reported, histone acetyltransferase A and DB, observed in calf thymus and bovine lymphocytes, respectively.

Purification and Properties of Bovine Pituitary Renin

1. Renin was purified to homogeneity from bovine anterior pituitary by using batchwise DEAE-cellulose chromatography, pepstatin-aminohexyl-agarose affinity chromatography, Ultrogel AcA 44 gel filtration and DEAE-Sephacel and CM-cellulose ion exchange chromatography. 2. The enzyme has a molecular weight of 36 000 and an isoelectric point of 5.25, and exhibits optimum activity at a pH between 6.5 and 7.5. 3. The amino acid composition and antigenic properties of this purified renin are very similar to those of rat, dog and hog kidney renins.

Purification and properties of a progesterone-induced plasmin/trypsin inhibitor from uterine secretions of pigs and its immunocytochemical localization in the pregnant uterus.

The porcine uterus secretes a group of basic, low molecular weight protease inhibitors under the influence of progesterone, but not estrogen. One of these inhibitors (Mr approximately 14,500) which inhibits trypsin, plasmin, and chymotrypsin, but not other proteases tested, has been purified 10- to 15-fold from uterine secretions of pseudopregnant pigs using Sephadex G-100 chromatography, CM-cellulose ion exchange chromatography, and Sephadex G-50 or Bio-Gel P-10 chromatography. The inhibitor which is relatively heat- and pH-stable forms a 1:1 molar complex with trypsin which is not dissociated in sodium dodecyl sulfate except by boiling. Chymotrypsin appears to bind at the same site on the inhibitor as trypsin. The inhibitor is high in half-cysteine residues and basic amino acids, and appears not to be a glycoprotein. Antiserum has been raised against the purified inhibitor in rabbits and used to test its distribution in pigs using the immunoperoxidase-staining technique on tissue sections. The inhibitor is associated only with the glandular and surface epithelium of the uterus. Endometrial explants from pseudopregnant animals, cultured in presence of L-[3H]leucine, release the inhibitor in radioactive form indicating that it is a uterine product. The antiserum against the inhibitor cross-reacts with at least three other, basic, low molecular weights plasmin/trypsin inhibitors in porcine uterine secretions, suggesting that a family of isoinhibitors exists which may constitute up to 15% of the protein in porcine uterine secretions. The inhibitor(s) appears to coat and to be taken up by the trophoectoderm cells of the elongating blastocyst during pregnancy. It is suggested that the inhibitors may serve to protect the uterus from proteases released by the porcine trophoblast or to prevent degradation of essential macromolecules, such as uteroferrin, which have to be taken up by the conceptus.

Biochemical characterisation of the Li locus, which controls the activity of the cyanogenic ?-glucosidase in Trifolium repens L.

The cyanogenic β-glucosidase (linamarase) was purified from white clover leaf tissue. The enzyme is a homodimer with a molecular weight of 105 300-103 400 daltons estimated from molecular exclusion chromatography. The effect of buffer ions on the pH optimum and charge properties of the enzyme are presented. A combination of molecular exclusion chromatography and CM cellulose ion exchange chromatography purified linamarase 16 fold to a single 62 000 dalton polypeptide on SDS polyacrylamide gel electrophoresis. This polypeptide represented about 5% of the total soluble leaf protein and can be seen as a prominent band in SDS polyacrylamide gel electrophoresis of crude leaf extracts from Li Li plants. Screening backcross progeny showed that extracts from li li plants, which have no linamarase activity, lack this 62 000 dalton polypeptide. Linamarase is the major glycoprotein in white clover leaf extracts which binds to Concanavalin A-Sepharose.

Purification and characterization of a gamma-melanotropin precursor from frozen human pituitary glands.

A new melanocyte-stimulating peptide has been isolated from acid extracts of frozen human pituitary glands by salt/ethanol fractionation, Sephadex G-75 gel filtration and DEAE- and cM-cellulose ion-exchange chromatography. The peptide is glycosylated, has an N-terminal tryptophan residue and an apparent mol.wt. of 16000 as estimated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Its amino acid analysis closely resembles residues Trp-105 to Gln-29 predicted for the common precursor protein of bovine corticotropin and beta-lipotropin by Nakanishi, Inoue, Kita, Nakamura, Chang, Cohen & Numa [(1979) Nature (London) 278, 423-427]. This fragment is expected to have melanotropin activity due to the tetrapeptide -His-Phe-Arg-Trp- (residues -51 to -48) of the predicted sequence of the common precursor. It was found to have a molar potency of 1 X 10(-5) relative to alpha-melanotropin in the frog skin bioassay. These characteristics are consistent with the isolated melanotropin peptide being a non-corticotropin, non-lipotropin peptide of the human common precursor protein of corticotropin and lipotropin. The peptide neither potentiates the adrenal weight-maintenance activity of corticotropin-(1-24)-tetracosapeptide when administered to hypophysectomized rats, nor stimulates release of non-esterified fatty acids from isolated rat epididymal cells. A second N-terminal-tryptophan glycopeptide was also isolated, which had an amino-acid composition similar to that predicted for the bovine common precursor protein, residues Trp-105 to Gly-35.

11] Isolation and characterization of Vitamin B12-binding proteins from human fluids

Publisher Summary This chapter discusses the isolation and characterization of vitamin B 12 -binding proteins from human fluids. Many different methods have been described for the measurement of vitamin B 12 -binding proteins in human serum. The techniques of protein separation that have been used include paper and starch–gel electrophoresis, Geon-block electrophoresis, column chromatography and immunodiffusion techniques of Ouchterlony, DEAE-cellulose and CM-cellulose ion-exchange chromatography, Sephadex G-200, affinity chromatography, and isoelectric focusing. The presence of at least 3 vitamin B 12 -binding proteins in human serum has been demonstrated. These binders have been designated as TC I, TC II, and TC III. This chapter describes the methods for isolation and characterization of B 12 -binding proteins from human serum. However, these methods can be used also for the isolation of binding proteins from human saliva, as well as other biological fluids. Gel filtration on Sephadex G-200 Column is described. Pattern of distribution of radioactivity of vitamin B 12 -binding proteins of normal human serum on a Sephadex G-200 column is illustrated. Pattern of distribution of radioactivity of vitamin B12 binders from human amniotic fluid on large DEAE-cellulose columns is also explained.