Trace elements, such as As, Co, Cr, Hg, Sb, and Zn, were determined by neutron activation analysis (NAA), whereas Cd, Cu, and Pb were determined by graphite furnace atomic absorption spectroscopy (GFAAS) in clam, crab, prawn, swamp cerith, and mussel samples after digestion by microwave heating under controlled conditions before eluting the solutions through a column of a chelating resin, Chelex-100. The standard used in the determination of percentage volatile elements retained by microwave digestion and also in the activation process was Lobster Hepatopancreas TORT-1, whereas known mixed standards were prepared from nitrate salts to determine the efficiency of the separation procedure at a controlled pH. Mercury and lead detected in crabs exceeded the maximum permissible level. Some species also showed a high affinity toward certain elements, and their levels of accumulation in the tissues of these species corresponded with the concentration of these elements in sediments, especially at sites in the vicinity of an industrial zone.
1. The L-amino acid oxidase of the monocellate cobra (Naja naja kaouthia) venom was purified to electrophoretic homogeneity. The molecular weight of the enzyme was 112,200 as determined by Sephadex G-200 gel filtration chromatography, and 57,400 as determined by SDS-polyacrylamide gel electrophoresis. 2. The enzyme had an isoelectric point of 8.12 and a pH optimum of 8.5. It showed remarkable thermal stability, and, unlike many venom L-amino acid oxidase, was also stable in alkaline medium. The enzyme was partially inactivated by freezing. 3. The enzyme was very active against L-phenylalanine and L-tyrosine, moderately active against L-tryptophan, L-methionine, L-leucine, L-norleucine, L-arginine and L-norvaline. Other L-amino acids were oxidized slowly or not oxidized. 4. Kinetic studies suggest the presence of a side-chain binding site in the enzyme, and that the binding site comprises of at least four hydrophobic subsites.
The fruit extracts of ripening cv. Harumanis mango contained a number of glycosidases and glycanases. Among the glycosidases, beta-D-galactosidase (EC 3.2.1.23) appeared to be the most significant. The enzyme activity increased in parallel with increase in tissue softness during ripening. Mango beta-galactosidase was fractionated into three isoforms, viz. beta-galactosidase I, II and III by a combination of chromatographic procedures on DEAE-Sepharose CL-6B, CM-Sepharose and Sephacryl S-200 columns. Apparent Km values for the respective beta-galactosidase isoforms for p-nitrophenyl beta-D-galactoside were 3.7, 3.3 and 2.7 mM, and their Vmax values were 209, 1024 and 62 nkat mg-1 protein. Optimum activity occurred at ca pH 3.2 for beta-galactosidase I and II, and pH 3.6 for beta-galactosidase III. Mango beta-galactosidase and its isoforms have galactanase activity, and the activity of the latter in the crude extracts generally increased during ripening. The close correlation between changes in beta-galactosidase activity, tissue softness, and increased pectin solubility and degradation suggests that beta-galactosidase might play an important role in cell wall pectin modification and softening of mango fruit during ripening.
1. An extract prepared from the tentacle of the jellyfish (CE), Catostylus mosaicus exhibited haemolytic, oedema and haemorrhage-inducing activities. 2. Acetone treatment of the tentacle extract produced an acetone soluble extract (AE) which showed an increase in specific haemolytic and haemorrhagic activities by 25- and 120-fold respectively; the minimum oedema dose was reduced by 30-fold. 3. The AE caused a rapid onset of oedema in the mouse foot pad. The effect was long-lasting, reaching a maximum in about 30 min after injection and sustained up to 4 hr. 4. Fractionation of the AE on Q-Sepharose gave 4 bound fractions which induced oedema and haemorrhage; however only 3 of the fractions exhibited haemolytic activity.
An acidic, lethal phospholipase Az was purified to electrophoretic homogeneity from the venom of the Malayan cobra (Naja naja sputatrix). The enzyme has an isoelectric point of 5.58, a molecular weight of 12000, and a medium lethal dose (LD50) of 0.86 micrograms/g in mice by intravenous injection. The enzyme also exhibited weak anticoagulant and edema-forming activities. The amino acid composition of the enzyme is similar to those of other cobra venom phospholipases Az.
Jackfruit is a sweet tropical fruit with very pleasant aroma, and the ripe seeds are edible. In this study, jackfruit seed proteins were isolated and subjected to trypsin digestion. The resultant protein hydrolysate was then subjected to antioxidant assay-guided purification, using centrifugal filtration, C18 reverse-phase and strong cation exchange (SCX) fractionations. The purified SCX fraction was further analyzed by de novo peptide sequencing, and two peptide sequences were identified and synthesized. Peptide JFS-2 (VGPWQK) was detected with antioxidant potential, with EC50 value comparable to that of commercial GSH antioxidant peptide. Additionally, the identified peptides were tested with protein protection potential, in an albumin protein denaturation inhibitory assay. Concurrently, we also investigated the pH, temperature, and gastrointestinal-digestion stability profiles for the identified peptide. With further research efforts, the identified peptides could potentially be developed into preservative agent for protein-rich food systems or as health-promoting diet supplements.
Medicinal mushrooms have been used for centuries against cancer and infectious diseases. These positive biological effects of mushrooms are due in part to the indirect action of stimulating immune cells. The objective of the current study is to investigate the possible immunomodulatory effects of mushroom polysaccharides on NK cells against different cancer cells. In this current study, fruiting bodies isolated from cultured Pleurotus ostreatus were extracted and partially purified using DEAE ion-exchange chromatography. The activation action of the collected fractions on Natural Killer cells was quantified against three different cancer cell lines in the presence or absence of human recombinant IL2 using three different activation and co-culture conditions. The possible modes of action of mushroom polysaccharides against cancer cells were evaluated at the cellular and molecular levels. Our results indicate that P. ostreatus polysaccharides induced NK-cells cytotoxic effects against lung and breast cancer cells with the largest effect being against breast cancer cells (81.2%). NK cells activation for cytokine secretion was associated with upregulation of KIR2DL genes while the cytotoxic activation effect of NK cells against cancer cells correlated with NKG2D upregulation and induction of IFNγ and NO production. These cytotoxic effects were enhanced in the presence of IL2. Analysis of the most active partially purified fraction indicates that it is predominantly composed of glucans. These results indicate bioactive 6-linked glucans present in P. ostreatus extracts activate NK-cell cytotoxicity via regulation of activation and induction of IFNγ and NO. These studies establish a positive role for bioactive P. ostreatus polysaccharides in NK-cells activation and induction of an innate immune response against breast and lung cancer cells.
Hepatitis B virus-like particles expressed in Escherichia coli were purified using anion exchange adsorbents grafted with polymer poly(oligo(ethylene glycol) methacrylate) in flow-through chromatography mode. The virus-like particles were selectively excluded, while the relatively smaller sized host cell proteins were absorbed. The exclusion of virus-like particles was governed by the accessibility of binding sites (the size of adsorbents and the charge of grafted dextran chains) as well as the architecture (branch-chain length) of the grafted polymer. The branch-chain length of grafted polymer was altered by changing the type of monomers used. The larger adsorbent (90 μm) had an approximately twofold increase in the flow-through recovery, as compared to the smaller adsorbent (30 μm). Generally, polymer-grafted adsorbents improved the exclusion of the virus-like particles. Overall, the middle branch-chain length polymer grafted on larger adsorbent showed optimal performance at 92% flow-through recovery with a purification factor of 1.53. A comparative study between the adsorbent with dextran grafts and the polymer-grafted adsorbent showed that a better exclusion of virus-like particles was achieved with the absorbent grafted with inert polymer. The grafted polymer was also shown to reduce strong interaction between binding sites and virus-like particles, which preserved the particles' structure.
Depolymerase is an enzyme that plays an important role in the hydrolysis of polyhydroxyalkanoates [PHAs]. In the current study, Burkholderia cepacia DP1 was obtained from Penang, Malaysia in which the enzyme was purified using ion exchange and gel filtration (Superdex-75) column chromatography. The molecular mass of the enzyme was estimated to be 53.3 kDa using SDS-PAGE. The enzyme activity was increased to 36.8 folds with the recovery of 16.3% after purification. The enzyme activity was detected between pH 6.0-10 and at 35-55 °C with pH 6.0 and 45 °C facilitating the maximum activity. Depolymerase was inactivated by Tween-20, Tween-80, SDS and PMSF, but insensitive to metal ions (Mg2+, Ca2+, K+, Na2+, Fe3+) and organic solvents (methanol, ethanol, and acetone). The apparent Km values of the purified P(3HB) depolymerase enzyme for P(3HB) and P(3HB-co-14%3HV) were 0.7 mg/ml and 0.8 mg/ml, respectively. The Vmax values of the purified enzyme were 10 mg/min and 8.89 mg/min for P(3HB) and P(3HB-co-14%3HV), respectively. The current study discovered a new extracellular poly(3-hydroxybutyrate) [P(3HB)] depolymerase enzyme from Burkholderia cepacia DP1 isolated and purified to homogeneity from the culture supernatant. To the best of our knowledge, this is the first report demonstrating the purification and biochemical characterization of P(3HB) depolymerase enzyme from genus Burkholderia.
Hepatitis B core antigen (HBcAg) is an important serological marker used in the diagnosis of hepatitis B virus (HBV) infections. In the current study, a fast and efficient preparative purification protocol for truncated HBcAg from Escherichia coli disruptate was developed. The recombinant HBcAg was first captured by anion exchange expanded bed adsorption chromatography integrated with a cell disruption process. This online capture process has shortened the process time and eliminated the "hold-up" period that may be detrimental to the quality of target protein. The eluted product from the expanded bed adsorption chromatography was subsequently purified using size-exclusion chromatography. The results showed that this novel purification protocol achieved a recovery yield of 45.1% with a product purity of 88.2%, which corresponds to a purification factor of 4.5. The recovered HBcAg is still biologically active as shown by ELISA test.
Matched MeSH terms: Chromatography, Ion Exchange/methods*
Poly(oligo(ethylene glycol) methacrylate) (POEGMA), an inert polymer was grafted onto an anion exchange adsorbent for the exclusion of relatively larger hepatitis B virus-like particles (HB-VLPs) from the anion exchange ligand (Q) and at the same time this process allowed the selective adsorption of smaller size Escherichia coli host cell proteins (HCPs). The chain lengths of the POEGMA grafted were modulated by varying the amount of monomers used in the polymer grafting. The purification factor and yield of the HB-VLPs obtained from the flow-through of negative chromatography were 2.3 and 66.0±3.1%, respectively, when shorter chain length of POEGMA (SQ) was grafted. Adsorbent grafted with longer chain of POEGMA (LQ) excluded some HCPs that are larger in size together with the HB-VLPs, reducing the purity of the recovered HB-VLPs. Further heat-treatment of the flow-through pool from SQ followed by centrifugation increased the purity of heat stable HB-VLPs to 87.5±1.1%. Heat-treatment of the flow through sample resulted in thermal denaturation and aggregation of HCPs, while the heat stable HB-VLPs still remained intact as observed under a transmission electron microscope. The performance of the negative chromatography together with heat treatment in the purification of HB-VLPs is far better than the reported bind-and-elute techniques.
Matched MeSH terms: Chromatography, Ion Exchange/methods
This paper describes a refinement in the purification step that facilitated the downstream recovery of high purity BmR1 recombinant protein, which is a protein used as a test reagent in the commercialized rapid tests for detection of lymphac filariasis i.e. Brugia Rapid™ and panLF rapid™. Purification was performed by immobilized metal affinity chromatography (IMAC), followed by ion exchange chromatography (IEX). Results showed that a total of 10.27 mg of BmR1 was obtained when IMAC was performed using 20 mM of imidazole and 5 column volume of wash buffer containing 500 mM of NaCl. Purity of the target protein was enhanced when buffer at pH 5.8 was used during the IEX. Two proteins that recurrently appeared below the BmR1 recombinant protein were identified by mass-spectrometry analysis as the same protein, thus they were probably degradation products of BmR1. These strategies improve purity of the target protein to be used in applications such as production of aptamers and monoclonal antibodies.
Matched MeSH terms: Chromatography, Ion Exchange/methods*
Enzymatic methods have been used to cleave the C- or N-terminus polyhistidine tags from histidine tagged proteins following expanded bed purification using immobilized metal affinity chromatography (IMAC). This study assesses the use of Factor Xa and a genetically engineered exopeptidase dipeptidyl aminopeptidase-1 (DAPase-1) for the removal of C-terminus and N-terminus polyhistidine tags, respectively. Model proteins consisting of maltose binding protein (MBP) having a C- or N-terminal polyhistidine tag were used. Digestion of the hexahistidine tag of MBP-His(6) by Factor Xa and HT15-MBP by DAPase-1 was successful. The time taken to complete the conversion of MBP-His(6) to MBP was 16 h, as judged by SDS-PAGE and Western blots against anti-His antibody. When the detagged protein was purified using subtractive IMAC, the yield was moderate at 71% although the overall recovery was high at 95%. Likewise, a yield of 79% and a recovery of 97% was obtained when digestion was performed with using "on-column" tag digestion. On-column tag digestion involves cleavage of histidine tag from polyhistidine tagged proteins that are still bound to the IMAC column. Digestion of an N-terminal polyhistidine tag from HT15-MBP (1 mg/mL) by the DAPase-I system was superior to the results obtained with Factor Xa with a higher yield and recovery of 99% and 95%, respectively. The digestion by DAPase-I system was faster and was complete at 5 h as opposed to 16 h for Factor Xa. The detagged MBP proteins were isolated from the digestion mixtures using a simple subtractive IMAC column procedure with the detagged protein appearing in the flowthrough and washing fractions while residual dipeptides and DAPase-I (which was engineered to exhibit a poly-His tail) were adsorbed to the column. FPLC analysis using a MonoS cation exchanger was performed to understand and monitor the progress and time course of DAPase-I digestion of HT15-MBP to MBP. Optimization of process variables such as temperature, protein concentration, and enzyme activity was developed for the DAPase-I digesting system on HT15-MBP to MBP. In short, this study proved that the use of either Factor Xa or DAPase-I for the digestion of polyhistidine tags is simple and efficient and can be carried out under mild reaction conditions.
Matched MeSH terms: Chromatography, Ion Exchange/methods
The proteome of Naja sumatrana (Equatorial spitting cobra) venom was investigated by shotgun analysis and a combination of ion-exchange chromatography and reverse phase HPLC. Shotgun analysis revealed the presence of 39 proteins in the venom while the chromatographic approach identified 37 venom proteins. The results indicated that, like other Asiatic cobra venoms, N. sumatrana contains large number of three finger toxins and phospholipases A2, which together constitute 92.1% by weight of venom protein. However, only eight of the toxins can be considered as major venom toxins. These include two phospholipases A2, three neurotoxins (two long neurotoxins and a short neurotoxin) and three cardiotoxins. The eight major toxins have relative abundance of 1.6-27.2% venom proteins and together account for 89.8% (by weight) of total venom protein. Other venom proteins identified include Zn-metalloproteinase-disintegrin, Thaicobrin, CRISP, natriuretic peptide, complement depleting factors, cobra venom factors, venom nerve growth factor and cobra serum albumin. The proteome of N. sumatrana venom is similar to proteome of other Asiatic cobra venoms but differs from that of African spitting cobra venom. Our results confirm that the main toxic action of N. sumatrana venom is neurotoxic but the large amount of cardiotoxins and phospholipases A2 are likely to contribute significantly to the overall pathophysiological action of the venom. The differences in toxin distribution between N. sumatrana venom and African spitting cobra venoms suggest possible differences in the pathophysiological actions of N. sumatrana venom and the African spitting cobra venoms, and explain why antivenom raised against Asiatic cobra venom is not effective against African spitting cobra venoms.
The L-amino acid oxidase (EC 1. 4. 3. 2) from King cobra (Ophiophagus hannah) venom was purified to electrophoretic homogeneity. The molecular weight of the enzyme was determined to be 140000 when examined by gel filtration and 68000 by SDS-polyacrylamide gel electrophoresis. The enzyme had an isoelectric point of 4.5 and an intravenous LD50 of 5 micrograms/g in mice. It is a glycoprotein and contains two moles of FAD per mole of enzyme. The enzyme exhibited unusual thermal stability and unlike most other venom L-amino acid oxidases, it was stable in alkaline solution and was not inactivated by freezing.
The toxic and biological activities of four samples of Trimeresurus purpureomaculatus venom were examined. The lethality, protein composition and biological activities of the four venom samples were similar. Three of the venom samples had LD50 (i.v.) values of 0.9 micrograms/g while the fourth had a lower LD50 (i.v.) of 0.45 micrograms/g. All four venom samples exhibited hemorrhagic, edema-inducing, anticoagulant and thrombin-like activities as well as the usual enzymes found in crotalid venoms. DEAE-Sephacel ion exchange chromatographic fractionation of the venom yielded 10 protein fractions. Only two fractions (fractions A and F) were lethal to mice; the major lethal fraction being fraction F. This fraction had an LD50 (i.v.) of 0.2 micrograms/g and exhibited hemorrhagic, edema-inducing and thrombin-like activity. It also exhibited phospholipase A, arginine ester hydrolase, arginine amidase, protease, 5'-nucleotidase, acetylcholinesterase and alkaline phosphomonoesterase activities. The lethal potency of fraction F is potentiated by fraction G, which exhibited anticoagulant activity as well as hemorrhagic, edema-inducing and enzymatic activities. Fractions F plus G account for almost 100% of the lethal potency of the venom.
Bungarus candidus venom exhibited high hyaluronidase, acetylcholinesterase and phospholipase A activities; low proteinase, 5'-nucleotidase, alkaline phosphomonoesterase and phosphodiesterase activities and moderately high L-amino acid oxidase activity. SP-Sephadex C-50 ion exchange chromatographic fractionation of the venom and Sephadex G-50 chromatography of the major lethal venom fractions indicate that the venom contains at least two highly lethal, basic phospholipases A with LD50 (i.v.) values of 0.02 micrograms/g (F6A) and 0.18 micrograms/g (F4A), respectively; as well as two polypeptide toxins with LD50 (i.v.) values of 0.17 micrograms/g and 0.83 micrograms/g, respectively. The major lethal toxin is the basic lethal phospholipase A, F6A, which accounts for approximately 13% of the venom protein and has a mol. wt of 21,000.
Sumatran pit viper (Trimeresurus sumatranus sumatranus) venom was fractionated by DEAE-Sephacel ion exchange chromatography into seven fractions. Fractions 4, 5 and 6 were lethal to mice and exhibited strong hemorrhagic activity, as well as some enzymatic activities. Fraction 6 also exhibited potent anticoagulant and thrombin-like activities. Analysis of the biological and enzymatic properties of the three lethal fractions suggests that the major lethal component of fractions 4 and 5 may be the hemorrhagic principle, and that the lethality of fraction 6 may be due to the hemorrhagic principle and/or the anticoagulant principle.
T1 Lipase is a thermostable secretary protein of Geobacillus zalihae strain previously expressed in a prokaryotic system and purified using three-step purification: affinity 1, affinity 2, and ion exchange chromatography (IEX). This approach is time consuming and offers low purity and recovery yield. In order to enhance the purification strategy of T1 lipase, affinity 2 was removed so that after affinity 1, the cleaved Glutathione S-transferase (GST) and matured T1 lipase could be directly separated through IEX. Therefore, a rational design of GST isoelectric point (pI) was implemented by prediction using ExPASy software in order to enhance the differences of pI values between GST and matured T1 lipase. Site-directed mutagenesis at two locations flanking the downstream region of GST sequences (H215R and G213R) was successfully performed. Double point mutations changed the charge on GST from 6.10 to 6.53. The purified lipase from the new construct GST tag mutant-T1 was successfully purified using two steps of purification with 6,849 U/mg of lipase specific activity, 33% yield, and a 44-fold increase in purification. Hence, the increment of the pI values in the GST tag fusion T1 lipase resulted in a successful direct separation through IEX and lead to successful purification.
Whole blood samples from patients with various forms of alpha- and beta- thalassaemia were incubated with 14C-Leucine to determine the relative rates of production of the alpha and beta chains by their reticulocytes. The labelled globin chains were fractionated by CM-Cellulose Chromatography in 8M Urea and the incorporated activity determined. The relative rates of synthesis of alpha and beta chains in some cases of alpha and beta- thalassaemia were established and the chain synthetic ratios were compared with similar ratios in normal individuals. The results show that it is possible to identify from the relative rates of in-vitro synthesis of the alpha and beta chains, the presence of the common thalassaemia slates in particular beta-thal trait, beta-thal homozygotes, Hb H disease and alpha0-thal trait. The presence of transfused blood does not affect the result. This study indicates that an abnormal alpha/beta chain synthesis ratio is useful in defining alpha and beta-thalassaemia variants.