Selective separation and sensitive detection of dissolved silicon and boron (DSi and DB) in aqueous solution was achieved by combining an electrodialytic ion isolation device (EID) as a salt remover, an ion-exclusion chromatography (IEC) column, and a corona charged aerosol detector (CCAD) in sequence. DSi and DB were separated by IEC on the H(+)-form of a cation exchange resin column using pure water eluent. DSi and DB were detected after IEC separation by the CCAD with much greater sensitivity than by conductimetric detection. The five-channel EID, which consisted of anion and cation acceptors, cathode and anode isolators, and a sample channel, removed salt from the sample prior to the IEC-CCAD. DSi and DB were scarcely attracted to the anion accepter in the EID and passed almost quantitatively through the sample channel. Thus, the coupled EID-IEC-CCAD device can isolate DSi and DB from artificial seawater and hot spring water by efficiently removing high concentrations of Cl(-) and SO4(2-) (e.g., 98% and 80% at 0.10molL(-1) each, respectively). The detection limits at a signal-to-noise ratio of 3 were 0.52μmolL(-1) for DSi and 7.1μmolL(-1) for DB. The relative standard deviations (RSD, n=5) of peak areas were 0.12% for DSi and 4.3% for DB.
Urease is a potent metalloenzyme with diverse applications. This paper describes the scale up and purification of an extracellular urease from Arthrobacter creatinolyticus MTCC 5604. The urease production was scaled-up in 3.7 L and 20 L fermentor. A maximum activity of 27 and 27.8 U/mL and a productivity of 0.90 and 0.99 U/mL/h were obtained at 30 h and 28 h in 3.7 and 20 L fermentor, respectively. Urease was purified to homogeneity with 49.85-fold purification by gel filtration and anion exchange chromatography with a yield of 36% and a specific activity of 1044.37 U/mg protein. The enzyme showed three protein bands with molecular mass of 72.6, 11.2 and 6.1 kDa on SDS-PAGE and ~ 270 kDa on native PAGE. The cytotoxic effect of urease was assessed in vitro using cancer cell lines (A549 and MG-63) and normal cell line (HEK 293). Urease showed its inhibitory effects on cancer cell lines through the generation of toxic ammonia, which in turn increased the pH of the surrounding medium. This increase in extracellular pH, enhanced the cytotoxic effect of weak base chemotherapeutic drugs, doxorubicin (50 µM) and vinblastine (100 µM) in the presence of urease (5 U/mL) and urea (0-4 mM) significantly.
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.
An anion exchange monolithic silica capillary column was prepared by surface modification of a hybrid monolithic silica capillary column prepared from a mixture of tetramethoxysilane (TMOS) and methyltrimethoxysilane (MTMS). The surface modification was carried out by on-column copolymerization of N-[3-(dimethylamino)propyl]acrylamide methyl chloride-quaternary salt (DMAPAA-Q) with 3-methacryloxypropyl moieties bonded as an anchor to the silica surface to form a strong anion exchange stationary phase. The columns were examined for their performance in liquid chromatography (LC) and capillary electrochromatography (CEC) separations of common anions. The ions were separated using 50 mM phosphate buffer at pH 6.6. Evaluation by LC produced an average of 30,000 theoretical plates (33 cm column length) for the inorganic anions and nucleotides. Evaluation by CEC, using the same buffer, produced enhanced chromatographic performance of up to ca. 90,000 theoretical plates and a theoretical plate height of ca. 4 mum. Although reduced efficiency was observed for inorganic anions that were retained a long time, the results of this study highlight the potential utility of the DMAPAA-Q stationary phase for anion separations.
A study was conducted to isolate, partial characterize Asian sea bass (Lates calcarifer) vitellogenin (vtg). Two-year-old juvenile L. calcarifer (n = 10) were given three intraperitoneal injections of 17-β estradiol (E2) at a dose of 2 mg/kg body weight to induce vitellogenesis. Blood was collected 3 days after the last injection, and plasma was purified through gel filtration chromatography. A broad single symmetrical peak consisting of vtg molecule was produced. Protein concentration was 0.059 mg/ml as determined by Bradfrod assay using bovine serum albumin as a standard. The protein appeared as one circulating form in Native PAGE considering the dimeric form of putative vtg with molecular weight of 545 kDa. In SDS-PAGE under reducing conditions, two major bands appeared at 232.86 and 118.80 kDa and minor bands at 100.60, 85.80 and 39.92 kDa, respectively. The purified vtg was used to generate a polyclonal antibody, and the specificity of antibody was assessed by Western blot analysis. Two major bands were immunoreacted, but no cross-reactivity was observed with plasma from non-induced males. The protein was characterized as phosphoglycolipoprotein as it positively stained for the presence of lipid, phosphorus and carbohydrate using Sudan Black B, methyl green and periodic acid/Schiff reagent solution, respectively. The amino acid composition was analyzed by high sensitivity amino acid analysis that showed high percentage of non-polar amino acids (~48 %). The results suggest the potential utilization of vtg as a basis tool to further study about reproductive physiology of this important economical species.
Mass production of fish broodstock with high quality eggs requires the knowledge on the chemical composition and physiochemical properties of vitellogenin (Vtg) during ovulation. Vtg is an egg yolk precursor phospholipoglycoprotein, and has been analysed to evaluate the reproductive conditions and determine the spawning period in captive and wild fish. In this study, Vtg was induced in male H. nemurus through three intramuscular injections of 17-estradiol (E2). The Vtg was purified from the serum using gel filtration chromatography and the purified protein was reduced via SDS-PAGE. One major polypeptide corresponding to 130 kDa was observed. Vtg identification was done using peptide mass fingerprint (PMF) from the trypsin digestion of male H. nemurus Vtg induced with E2. The sequence homology of H. nemurus AYLAGAAADVLEVGVR matched the Vtg of other fish species when analysed using MALDI-TOF. Vtg was confirmed by MASCOT at 95% significant level. The potential protein that controls the reproductive process and oocyte development isolated from this study was discussed to understand the structure and function of Vtg.
Globally, peptide-based anticancer therapies have drawn much attention. Marine organisms are a reservoir of anticancer peptides that await discovery. In this study, we aimed to identify cytotoxic oligopeptides from Sarcophyton glaucum. Peptides were purified from among the S. glaucum hydrolysates produced by alcalase, chymotrypsin, papain, and trypsin, guided by a methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay on the human cervical cancer (HeLa) cell line for cytotoxicity evaluation. Purification techniques adopted were membrane ultrafiltration, gel filtration chromatography, solid phase extraction (SPE), and reversed-phase high-performance liquid chromatography (RP-HPLC). Purified peptides were identified by de novo peptide sequencing. From papain hydrolysate, three peptide sequences were identified: AGAPGG, AERQ, and RDTQ (428.45, 502.53, and 518.53 Da, respectively). Peptides synthesized from these sequences exhibited cytotoxicity on HeLa cells with median effect concentration (EC50) values of 8.6, 4.9, and 5.6 mmol/L, respectively, up to 5.8-fold stronger than the anticancer drug 5-fluorouracil. When tested at their respective EC50, AGAPGG, AERQ, and RDTQ showed only 16%, 25%, and 11% cytotoxicity to non-cancerous Hek293 cells, respectively. In conclusion, AERQ, AGAPGG, and RDTQ are promising candidates for future development as peptide-based anticancer drugs.
Macrobrachium rosenbergii nodavirus (MrNV) is the causative agent of white tail disease (WTD) which seriously impedes the production of the giant freshwater prawn and has a major economic impact. MrNV contains two segmented RNA molecules, which encode the RNA dependent RNA polymerase (RdRp) and the capsid protein (MrNV-CP) containing 371 amino acid residues. MrNV-CP comprises of the Shell (S) and the Protruding (P) domains, ranging from amino acid residues 1-252 and 253-371, respectively. The P-domain assembles into dimeric protruding spikes, and it is believed to be involved in host cell attachment and internalization. In this study, the recombinant P-domain of MrNV-CP was successfully cloned and expressed in Escherichia coli, purified with an immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC) up to ~90% purity. Characterization of the purified recombinant P-domain with SEC revealed that it formed dimers, and dynamic light scattering (DLS) analysis demonstrated that the hydrodynamic diameter of the dimers was ~6 nm. Circular dichroism (CD) analysis showed that the P-domain contained 67.9% of beta-sheets, but without alpha-helical structures. This is in good agreement with the cryo-electron microscopic analysis of MrNV which demonstrated that the P-domain contains only beta-stranded structures. Our findings of this study provide essential information for the production of the P-domain of MrNV-CP that will aid future studies particularly studies that will shed light on anti-viral drug discovery and provide an understanding of virus-host interactions and the viral pathogenicity.
In recent years, there are growing trends in using palm oil as raw materials in radiation curable resins production. In this study, the acrylated palm oil resins i.e. the EPOLA (epoxidized palm oil acrylate) and the POBUA (palm oil based urethane acrylate) were synthesized using two different systems, i.e. the 25 liter pilot scale reactor synthesis system and the 2 liter (L) laboratory scale reactor synthesis system through chemical processes known as acrylation and isocyanation. In this
paper, the property of the acrylated resins which were produced by these two systems were evaluated and compared between each other. Their properties were characterized using the Fourier transform infrared (FTIR) spectrophotometer for functional group identification; the gel permeation chromatography (GPC) for molecular weight (Mw) determination, the Brookfield viscometer for viscosity measurements, the acid values (AV) and the oxirane oxygen contents (OOC) analysis. As a result, the production process for both the 2 L and 25 L reactor system were found to be time consuming and the main advantages for the 25 L reactor was its higher productivity as compared with the 2 L reactor system with the same synthesis process parameters i.e. the temperatures and the experimental methods. Besides that, the 25 L reactor synthesis
process was found to be safe, easy to control and served unpolluted process to the environments. The final products, the acrylated palm oil resins were formulated into ultraviolet (UV) curable compounds before subjecting them under UVirradiation. As a result, the UV-curable palm oil resins showed potential uses as pressure sensitive adhesives, printing inks including overprint varnishes (OPV) and coatings.
Protein drugs are important therapeutic agents however; they may degrade during formulation processing. The objective of this study was to investigate the correlation between secondary structure alterations and the retentions of biological activity of protein upon the application of thermal stress. Catalase, horseradish peroxidase and α- chymotrypsin were employed as model proteins. Each protein was heated in a solid and solution state at a temperature of 70 °C for 1 h. Attenuated total reflectance Fourier transform infrared spectroscopy, size-exclusion chromatography and biological activity assay were performed. Results showed that heat-exposure of protein solids at 70 °C caused minimum changes in secondary structure and biological activity was almost retained. However, thermal exposure of protein aqueous solution induced significant changes in the secondary structure indicated by area overlap values and caused considerable reduction in the biological activity. The changes in secondary structures were found to be in full alignment with the loss of biological activity for both protein solids as well as aqueous solutions. Catalase lost entire biological activity upon heating in the solution state. In conclusion, the findings of the present study indicate a direct correlation between protein secondary structure alterations and the retention of biological activity which can be taken into account during the development and delivery of protein drugs formulations.
Bevacizumab is a full-length human monoclonal antibody used to treat various neovascular diseases such as wet age-related macular degeneration (AMD), diabetic eye disease and other problems of the retina. Monthly intravitreal injections of bevacizumab (Avastin®) are effective in the treatment of wet AMD. However, there is a growing demand in the development of sustained release ophthalmic formulations. Therefore, this study aims, for the first time, to develop a rapid, simple, and sensitive method using size exclusion chromatography coupled with fluorescence detection for routine quantification of bevacizumab in ophthalmic formulations and during in vitro release studies. The selected chromatographic conditions included an aqueous mobile phase composed of 35 mM sodium phosphate buffer and 300 mM sodium chloride (pH 6.8), a flow rate of 0.5 mL/min, and the fluorescence detector was operated at excitation and emission wavelengths of 280 and 340 nm, respectively. The peak area-concentration relationship maintained its linearity over concentration range of 0.1-20 μg/mL (R2 = 0.9993), and the quantitation limit was 100 ng/mL. The method was validated for specificity, accuracy, precision, and robustness. The developed method had a run time of 6 min at temperature 25 °C, making it a unique validated method for rapid and cost-effective quantification of bevacizumab.
Extracts from ‘kesinai’ (Streblus asper) leaves were investigated as a potential source of enzymes that can serve as an alternative to calf rennet in cheese making. Different types of extraction buffers were investigated namely sodium acetate buffer (pH 4.2-5.0), phosphate buffer (pH 6.0-7.0) and Tris-HCl buffer (pH 7.0-9.0). Finally, the milk-clotting enzyme was extracted using 100 mM Tris-HCl buffer (pH 7.4) with and without 5.0 mg/mL polyvinylpyrrolidone, 0.015 mL/mL Triton X-100 and 2 mM sodium metabisulphite. Purification was carried out using acetone precipitation, and ion-exchange and size-exclusion chromatographic techniques. Results showed that 100 mM Tris-HCl buffer (pH 7.4) was the most efficient extraction buffer among the buffers used in the extraction study. After the final purification step of size-exclusion chromatography, the enzyme was purified 3.3-fold with 42.3% of recovery. The enzyme showed an optimum temperature and pH at 60°C and pH 7.4, respectively. The enzyme was stable up to 70°C for one hour and the partially purified enzyme retained 83% and 96% of its original activity at pH 6.0 and 8.0, respectively. The molecular weight of the partially enzyme was estimated to be 75.8 kDa on SDS-PAGE. The milk-clotting activity of ‘kesinai’ enzyme was found to be lower than that of commercial Mucor rennet.
Recently, surface engineered biomaterials through surface modification are extensively investigated due to its potential to enhance cellular homing and migration which contributes to a successful drug delivery process. This study is focused on osteoblasts response towards surface engineered using a simple sodium hydroxide (NaOH) hydrolysis and growth factors conjugated poly(lactic acid) (PLA) microspheres. In this study, evaluation of the relationship of NaOH concentration with the molecular weight changes and surface morphology of PLA microspheres specifically wall thickness and porosity prior to in vitro studies was investigated. NaOH hydrolysis of 0.1 M, 0.3 M and 0.5 M were done to introduce hydrophilicity on the PLA prior to conjugation with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Morphology changes showed that higher concentration of NaOH could accelerate the hydrolysis process as the highest wall thickness was observed at 0.5 M NaOH with ~3.52 µm. All surface modified and growth factors conjugated PLA microspheres wells enhanced the migration of the cells during wound healing process as wound closure was 100% after 3 days of treatment. Increase in hydrophilicity of the surface engineered and growth factors conjugated PLA microspheres provides favorable surface for cellular attachment of osteoblast, which was reflected by positive DAPI staining of the cells' nucleus. Surface modified and growth factors conjugated PLA microspheres were also able to enhance the capability of the PLA in facilitating the differentiation process of mesenchymal stem cells (MSCs) into osteogenic lineage since only positive stain was observed on surface engineered and growth factors conjugated PLA microspheres. These results indicated that the surface engineered and growth factors conjugated PLA microspheres were non-toxic for biological environments and the improved hydrophilicity made them a potential candidate as a drug delivery vehicle as the cells can adhere, attach and proliferate inside it.
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.
The discovery of jacalin, a group of lectins from jackfruit seeds (Artocarpus heterophyllus), has attracted considerable attention due to its numerous interesting immunological properties as well as its usefulness in the isolation of various serum proteins. We have further identified a similar lectin from the seeds of Champedak (Artocarpus integer) which we refer to as lectin-C and performed comparative studies with two types of jacalin isolated from different batches of the Malaysian jackfruit seeds (jacalin-M1 and jacalin-M2). The three purified lectins demonstrated equivalent apparent Mr of about 52,500, each of which comprised of a combination of two types of non-covalently-linked subunits with apparent Mr of approximately 13,300 and 16,000. The lectins demonstrated equal haemagglutinating activity against human erythrocytes of blood groups A, B, AB and O. Our data also demonstrated that lectin-C, jacalin-M1 and jacalin-M2 are similar by selectively precipitating human serum IgA1 and colostral sIgA but not IgA2, IgD, IgG and IgM. When immunoelectrophoresis was performed on normal human sera and reacted with the lectins, single precipitin arcs corresponding to IgA immunoprecipitates were detected with lectin-C and jacalin-MI. Jacalin-M2, however, exhibited two closely associated precipitin arcs. The binding of these lectins with IgA was pronouncedly inhibited in the presence of p-nitrophenyl-beta-D-galactopyranoside, 1-o-methyl-alpha-D-galactopyranoside, D-melibiose, N-acetyl-D-galactosamine and D-galactose. The data therefore provide evidence on the differential specificity of IgA binding lectins isolated from seeds of similar as well as distinct Artocarpus species.
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.
1. The hemorrhagic, procoagulant, anticoagulant, phosphodiesterase, alkaline phosphomonoesterase, 5'-nucleotidase, hyaluronidase, arginine ester hydrolase, phospholipase A, L-amino acid oxidase and protease activities of 26 samples of venoms from 13 species of Bothrops were determined, and the Sephadex G-75 gel filtration patterns for some of the venoms also examined. 2. The results show that while there are considerable individual variations in the biological activities of many of the Bothrops venoms tested, there are some common characteristics at the genus and species levels. 3. The differences in the biological properties of the Bothrops venoms tested can be used for the differentiation of most Bothrops species examined.
1. Glutathione transferases from the liver, lung and kidney tissues of the buffalo (Bubalus bubalis) and the Kedah-Kelantan cattle (Bos indicus) were partially purified by ammonium sulphate precipitation and Sephadex G-75 gel filtration. 2. Liver tissue contains the highest enzyme activity when compared to the lung and kidney tissues. 3. The activity in cattle is higher than that in the buffalo. 4. Isoelectric focusing separates the activities into the acidic, near neutral and basic fractions. 5. The focused patterns are different for each of the tissues and in each of the species investigated.
The specificity and sensitivity of an indirect and two (an 'ordinary' and a 'rapid') double sandwich enzyme-linked immunosorbent assay (ELISA) procedures for the quantitation of Calloselasma rhodostoma (Malayan pit viper) venom were examined. The three assays were equally sensitive and the accuracy of the assays was not substantially affected by individual variation in the venom composition. The specificity of the assays was examined against 26 venoms from snakes of the families Viperidae and Elapidae. While the double sandwich ELISA procedures were sufficiently specific to be used in the clinical immunodiagnosis of C. rhodostoma bite in Malaysia, the indirect ELISA procedure exhibited extensive cross-reactivity with other Malaysian pit viper venoms. Attempts were made to improve the specificity of the indirect ELISA procedure for the quantitation of C. rhodostoma venom. A 'low ELISA cross-reactivity' venom fraction (termed VF52) was isolated from C. rhodostoma venom by repeated Sephadex G-100 gel filtration chromatography. The indirect ELISA procedure using antibodies to VF52 as immunoreagent showed an improvement in specificity. The use of the indirect ELISA procedure for the detection of C. rhodostoma antibodies was also examined and the results show that the assay was sufficiently specific to be used for retrospective diagnosis of C. rhodostoma bite in Malaysia, in particular when VF52 was used as the coating antigen.
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.