Hydroxyapatite Column Chromatography Research Articles

Adverse effects of 2,4-D dimethylammonium based-herbicide on Acetylcholinesterase expression in Nile tilapia (Oreochromis niloticus)

In this study, the evaluation of a 2,4-D dimethylammonium based-herbicide impacted on Nile tilapia was done. The effects focus on Acetylcholinesterase (AChE) expression in the brain, gill, muscle, and plasma using antibody techniques. Our findings revealed a decrease in AChE expression with prolonged exposure. For these, AChE was purified using hydroxyapatite column chromatography. Moreover, the isolated protein was characterized as AChE by Polyclonal Ab specific to AChE through the Western blot. For interpretation at the cellular and molecular level, we employed two analytical techniques, histology, and optical coherence tomography (OCT). Alterations in the gill, liver, and muscle were observed to increase with increased exposure time. Field study concludes that AChE could serve as a biomarker to detect herbicide contamination in water and its accumulation in aquatic animals. This study may aid in surveillance and strategy formulation for managing contamination from such substances in various water sources.

Purification of Tomato Bushy Stunt Virus Particles by One-Step Hydroxyapatite Column Chromatography

The main aim of this work was to develop a time-saving and cost-effective purification method of infectious plant viral nanoparticles. Virions of Tomato bushy stunt virus (TBSV), which is a member of Tombusvirus genus, were purified by one-step Bio-gel HT Hydroxyapatite (HA) column chromatography. Extracts from Nicotiana benthamiana plants infected with TBSV were directly loaded onto the HA column and eluted by 10 mM sodium phosphate buffer (pH 6.8). A specificity of virions has been confirmed by immunoblotting and electron microscopy. Homogeneity of virions was tested by SDS-PAGE, where only 41 kDa polypeptide bands referring to the capsid protein of TBSV were detected by Coomassie staining. The biological infectious activity of a purified material was demonstrated by observing TBSV-specific symptoms observed in N. benthamiana plants at 7‒10 days of post-inoculation (dpi). Moreover, purified virions were used for immunization of the BALb/c mouse to raise primary antibodies against the TBSV virus. Our results show that in low concentrations of sodium phosphate buffer total proteins extracted from infected plants adsorb to HA sorbent, while viral particles do not adsorb to the HA matrix and flow throw column due to Ca2+ ions implicated in TBSV virions’ structure. This highly effective and simple virus purification protocol can also be used for the isolation of other plant virions.

Effective refolding of a cysteine rich glycoside hydrolase family 19 recombinant chitinase from Streptomyces griseus by reverse dilution and affinity chromatography.

Conventional refolding methods are associated with low yields due to misfolding and high aggregation rates or very dilute proteins. In this study, we describe the optimization of the conventional methods of reverse dilution and affinity chromatography for obtaining high yields of a cysteine rich recombinant glycoside hydrolase family 19 chitinase from Streptomyces griseus HUT6037 (SgChiC). SgChiC is a potential biocontrol agent and a reference enzyme in the study and development of chitinases for various applications. The overexpression of SgChiC was previously achieved by periplasmic localization from where it was extracted by osmotic shock and then purified by hydroxyapatite column chromatography. In the present study, the successful refolding and recovery of recombinant SgChiC (r-SgChiC) from inclusion bodies (IB) by reverse dilution and column chromatography methods is respectively described. Approximately 8 mg of r-SgChiC was obtained from each method with specific activities of 28 and 52 U/mg respectively. These yields are comparable to that obtained from a 1 L culture volume of the same protein isolated from the periplasmic space of E. coli BL21 (DE3) as described in previous studies. The higher yields obtained are attributed to the successful suppression of aggregation by a stepwise reduction of denaturant from high, to intermediate, and finally to low concentrations. These methods are straight forward, requiring the use of fewer refolding agents compared with previously described refolding methods. They can be applied to the refolding of other cysteine rich proteins expressed as inclusion bodies to obtain high yields of actively folded proteins. This is the first report on the recovery of actively folded SgChiC from inclusion bodies.

Acetylcholinesterase (AChE) polyclonal antibody from hybrid catfish (C. macrocephalus × C. gariepinus): Specification, sensitivity and cross reactivity

AChE (acetylcholinesterase) is generally classified as a specific biomarker of pesticide exposure. The aim of this study was to produce AChE polyclonal antibody from hybrid catfish that were exposed to commercial glyphosate. The hybrid catfish was exposed to glyphosate (0.75 mL/L) for 24 h. After that, the fish brain was dissected, AChE was extracted and purified by hydroxyapatite column chromatography and eluted with 0.2 M potassium phosphate buffer pH 6.8. This protocol gave 70% yield. Then, the brain extract was characterized using 10% SDS-PAGE and Western blot probed with commercial polyclonal antibody specific to AChE (PAb-AChE). The protein, 71 kDa, was then used as an antigen to immunize mice for antibody production. The polyclonal antibody (PAb) was characterized using dot blot, Western blot and immunohistochemistry for immunolocalization of AChE in hybrid catfish exposed to glyphosate. We found that the appropriate dilution of antibody for both dot blot and Western blot was 1:3500, and 1:2500 for immunohistochemistry. Cross reactivity testing showed that PAb-AChE can be used with AChE from striped snakehead fish at the same dilution as used with AChE from hybrid catfish. It was concluded that PAb specific to hybrid catfish AChE from this work was highly specific and sensitive, and can cross-react with striped snakehead fish AChE. Thus, this polyclonal antibody may be used in monitoring glyphosate exposure in hybrid catfish and striped snakehead fish.

Purification and characterization of adenovirus core protein VII: a histone-like protein that is critical for adenovirus core formation.

Adenovirus protein VII is a highly cationic core protein that forms a nucleosome-like structure in the adenovirus core by condensing DNA in combination with protein V and mu. It has been proposed that protein VII could condense DNA in a manner analogous to mammalian histones. Due to the lack of an expression and purification protocol, the interactions between protein VII and DNA are poorly understood. In this study we describe methods for the purification of biologically active recombinant protein VII using an E. coli expression system. We expressed a cleavable fusion of protein VII with thioredoxin and established methods for purification of this fusion protein in denatured form. We describe an efficient method for resolving the cleavage products to obtain pure protein VII using hydroxyapatite column chromatography. Mass spectroscopy data confirmed its mass and purity to be 19.4 kDa and >98 %, respectively. Purified recombinant protein VII spontaneously condensed dsDNA to form particles, as shown by dye exclusion assay, electrophoretic mobility shift assay and nuclease protection assay. Additionally, an in vitro bioluminescence assay revealed that protein VII can be used to enhance the transfection of mammalian cells with lipofectamine/DNA complexes. The availability of recombinant protein VII will facilitate future studies of the structure of the adenovirus core. Improved understanding of the structure and function of protein VII will be valuable in elucidating the mechanism of adenoviral DNA condensation, defining the morphology of the adenovirus core and establishing the mechanism by which adenoviral DNA enters the nucleus.

The hyperthermophilic cystathionine γ-synthase from the aerobic crenarchaeon Sulfolobus tokodaii: expression, purification, crystallization and structural insights.

Cystathionine γ-synthase (CGS; EC 2.5.1.48), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyzes the formation of cystathionine from an L-homoserine derivative and L-cysteine in the first step of the transsulfuration pathway. Recombinant CGS from the thermoacidophilic archaeon Sulfolobus tokodaii (StCGS) was overexpressed in Escherichia coli and purified to homogeneity by heat treatment followed by hydroxyapatite and gel-filtration column chromatography. The purified enzyme shows higher enzymatic activity at 353 K under basic pH conditions compared with that at 293 K. Crystallization trials yielded three crystal forms from different temperature and pH conditions. Form I crystals (space group P21; unit-cell parameters a = 58.4, b = 149.3, c = 90.2 Å, β = 108.9°) were obtained at 293 K under acidic pH conditions using 2-methyl-2,4-pentanediol as a precipitant, whereas under basic pH conditions the enzyme crystallized in form II at 293 K (space group C2221; unit-cell parameters a = 117.7, b = 117.8, c = 251.3 Å) and in form II' at 313 K (space group C2221; unit-cell parameters a = 107.5, b = 127.7, c = 251.1 Å) using polyethylene glycol 3350 as a precipitant. X-ray diffraction data were collected to 2.2, 2.9 and 2.7 Å resolution for forms I, II and II', respectively. Structural analysis of these crystal forms shows that the orientation of the bound PLP in form II is significantly different from that in form II', suggesting that the change in orientation of PLP with temperature plays a role in the thermophilic enzymatic activity of StCGS.

Purification and enzymatic characterization of membrane-bound d-gluconate dehydrogenase from Arthrobacter globiformis

Arthrobacter globiformis C224 is an industrial 2-keto-gluconic acid (2KGA) producer and currently used for erythorbic acid production in China. In the present study, a membrane-bound gluconate dehydrogenase (GADH) with specific activity of 326.06U/mg and purification fold of 412 was purified from A. globiformis using a five-step procedure including ultrasonication, phase separation with Triton X-114, ammonium sulfate fractionation, DEAE-sepharose fast flow and hydroxyapatite column chromatography. The GADH was identified as to be flavin adenine dinucleotide (FAD)-dependent and consisted of three subunits with molecular mass of 66,000Da, 44,000Da and 23,000Da. The optimal pH and temperature of A. globiformis GADH were 5.0 and 40°C, respectively. It had the stable activity at pH of 5.0–7.0 or below 50°C, and the strict substrate specificity for d-gluconate with the Km of 3.15mmolL−1, 1.04mmolL−1 at pH 5.0 and pH 6.0, respectively. The GADH activity also was significantly influenced by metal ions, organic solvents, and organic acids. Our study will benefit for better understanding of 2KGA production process by A. globiformis C224.

Evidence for the binding affinity of daunomycin to HMGB1 protein in chromatin and in solution

In this study the interaction of daunomycin with HMGB1 nonhistone chromatin protein in the chromatin context using hydroxyapatite (HAP) column chromatography and free in solution was investigated employing fluorescence, circular dichroism spectroscopy and thermal denaturation techniques. The results demonstrate that HMGB1 fraction eluted from HAP column contained the most amount of daunomycin. Upon addition of daunomycin to HMGB1 solution, fluorescence emission intensity was dependent on the drug concentration used whereas the ellipticity in CD spectra was decreased at both 205 and 220nm extremes implying that quenching of the drug with the HMGB1 chromospheres alters secondary structure of the protein. Although daunomycin slightly increased the melting point of HMGB1, but exhibited a significant hyperchromicity at low concentrations and hypochromicity at higher concentrations of daunomycin. The results suggest that daunomycin binds to HMGB1 protein which may influence its interaction with DNA in nucleosomes and other cellular processes.

A novel cold-adapted lipase, LP28, from a mesophilic Streptomyces strain

Fossil fuel is limited but its usage has been growing rapidly, thus the fuel is predicted to be completely running out and causing an unbearable global energy crisis in the near future. To solve this potential crisis, incorporating with increasing environmental concerns, significant attentions have been given to biofuel production in the recent years. With the aim of isolating a microbial biocatalyst with potential application in the field of biofuel, a lipase from Streptomyces sp. CS628, LP28, was purified using hydroxyapatite column chromatography followed by a gel filtration. Molecular weight of LP28 was estimated to be 32,400 Da by SDS-PAGE. The activity was the highest at 30 °C and pH 8.0 and was stable at pH 6.0-8.0 and below 25 °C. The enzyme preferentially hydrolyzed p-nitrophenyl decanoate (C10), a medium chain substrate. Furthermore, LP28 non-specifically hydrolyzed triolein releasing both 1,2- and 1,3-diolein. More importantly, LP28 manifestly catalyzed biodiesel production using palm oil and methanol; therefore, it can be a potential candidate in the field of biofuel.

Identification of Glycan Structure Alterations on Cell Membrane Proteins in Desoxyepothilone B Resistant Leukemia Cells

Resistance to tubulin-binding agents used in cancer is often multifactorial and can include changes in drug accumulation and modified expression of tubulin isotypes. Glycans on cell membrane proteins play important roles in many cellular processes such as recognition and apoptosis, and this study investigated whether changes to the glycan structures on cell membrane proteins occur when cells become resistant to drugs. Specifically, we investigated the alteration of glycan structures on the cell membrane proteins of human T-cell acute lymphoblastic leukemia (CEM) cells that were selected for resistance to desoxyepothilone B (CEM/dEpoB). The glycan profile of the cell membrane glycoproteins was obtained by sequential release of N- and O-glycans from cell membrane fraction dotted onto polyvinylidene difluoride membrane with PNGase F and β-elimination respectively. The released glycan alditols were analyzed by liquid chromatography (graphitized carbon)-electrospray ionization tandem MS. The major N-glycan on CEM cell was the core fucosylated α2-6 monosialo-biantennary structure. Resistant CEM/dEpoB cells had a significant decrease of α2-6 linked sialic acid on N-glycans. The lower α2-6 sialylation was caused by a decrease in activity of β-galactoside α2-6 sialyltransferase (ST6Gal), and decreased expression of the mRNA. It is clear that the membrane glycosylation of leukemia cells changes during acquired resistance to dEpoB drugs and that this change occurs globally on all cell membrane glycoproteins. This is the first identification of a specific glycan modification on the surface of drug resistant cells and the mechanism of this downstream effect on microtubule targeting drugs may offer a route to new interventions to overcome drug resistance.

Purification, crystallization and preliminary X-ray analysis of phycocyanin and phycoerythrin from Porphyra yezoensis Ueda.

Porphyra yezoensis is one of the most important and widely cultured seaweeds in China. Phycobiliproteins exhibit excellent spectroscopic properties and play versatile roles in the biomedical, food, cosmetics and chemical synthetic dye industries. Here, the purification and crystallization of phycoerythrin and phycocyanin, two phycobiliproteins extracted from P. yezoensis, are described. Using a novel protocol including co-precipitation with ammonium sulfate and hydroxyapatite column chromatography, both phycobiliproteins were produced on a large scale with improved quality and yield compared with those previously reported. Native PAGE analysis indicated that phycoerythrin and phycocyanin exist as (αβ)(3) heterohexamers in solution. The crystals of phycoerythrin diffracted to 2.07 Å resolution and belonged to space group R3. The unit-cell parameters referred to hexagonal axes are a = b = 187.7, c = 59.7 Å, with nine (αβ)(2) heterotetramers per unit cell. The crystals of phycocyanin diffracted to 2.70 Å resolution in space group P2(1). Matthews coefficient analysis shows that 10-19 (αβ) heterodimers of phycocyanin in the asymmetric unit would be reasonable. A self-rotation function calculation clarified this ambiguity and indicated that 12 (αβ) heterodimers of phycocyanin are assembled in the asymmetric unit.

Sulfoacetate Is Degraded via a Novel Pathway Involving Sulfoacetyl-CoA and Sulfoacetaldehyde in Cupriavidus necator H16

Bacterial degradation of sulfoacetate, a widespread natural product, proceeds via sulfoacetaldehyde and requires a considerable initial energy input. Whereas the fate of sulfoacetaldehyde in Cupriavidus necator (Ralstonia eutropha) H16 is known, the pathway from sulfoacetate to sulfoacetaldehyde is not. The genome sequence of the organism enabled us to hypothesize that the inducible pathway, which initiates sau (sulfoacetate utilization), involved a four-gene cluster (sauRSTU; H16_A2746 to H16_A2749). The sauR gene, divergently orientated to the other three genes, probably encodes the transcriptional regulator of the presumed sauSTU operon, which is subject to inducible transcription. SauU was tentatively identified as a transporter of the major facilitator superfamily, and SauT was deduced to be a sulfoacetate-CoA ligase. SauT was a labile protein, but it could be separated and shown to generate AMP and an unknown, labile CoA-derivative from sulfoacetate, CoA, and ATP. This unknown compound, analyzed by MALDI-TOF-MS, had a relative molecular mass of 889.7, which identified it as protonated sulfoacetyl-CoA (calculated 889.6). SauS was deduced to be sulfoacetaldehyde dehydrogenase (acylating). The enzyme was purified 175-fold to homogeneity and characterized. Peptide mass fingerprinting confirmed the sauS locus (H16_A2747). SauS converted sulfoacetyl-CoA and NADPH to sulfoacetaldehyde, CoA, and NADP(+), thus confirming the hypothesis.

Use of hydroxyapatite-column chromatography combined with mass spectrometric analysis to assess the relative bone mineral-binding affinities of bisphosphonates at different PHS

Use of hydroxyapatite-column chromatography combined with mass spectrometric analysis to assess the relative bone mineral-binding affinities of bisphosphonates at different PHS

Determination of the relative bone mineral-binding affinities of bisphosphonates by using hydroxyapatite-column chromatography combined with mass spectrometric analysis

Determination of the relative bone mineral-binding affinities of bisphosphonates by using hydroxyapatite-column chromatography combined with mass spectrometric analysis

Designed mutants of human farnesyl pyrophosphate synthase (FPPS) help to characterise the interactions of nitrogen-containing bisphosphonates with Tyrosine 204 and Phenylalanine 239 side-chain residues

Determination of the relative bone mineral-binding affinities of bisphosphonates by using hydroxyapatite-column chromatography combined with mass spectrometric analysis Xuchen Duan, Zhidao Xia, Hao Zhang, Mike Quijano, Roy Dobson, Bobby Barnett, James Triffitt, James Dunford, Frank Ebetino, R. Graham Russell Nuffield Department of Orthopaedics, Rheumatology & Musculoskeletal Sciences, The Botnar Research Centre, Oxford University Institute of Musculoskeletal Sciences, Oxford, United Kingdom Procter & Gamble, Mason, Ohio, United States Department of Chemistry, University of Cincinnati, Cincinnati, Ohio, United States Warner Chilcott, Mason, Ohio, United States

Similarities and differences in the properties of multiple NDP-kinase isoforms of Alyssum murale, Ni 2+-accumulator species

Two isoforms of NDPKs (diphosphonucleoside kinases: E.C. 2.7.4.6.) named S-NDPK-A and S-NDPK-B were separated and purified from shoots of Alyssum murale (19th day of growth), a nickel accumulator plant, by a four-step procedure involving ammonium sulphate precipitation and DEAE-sepharose and hydroxyapatite column chromatography. Shoot NDPKs underwent autophosphorylation, proved thermostable, displayed similar molecular mass of 105,000, and consisted of six catalytic subunits. The size of subunits of S-NDPK-A and S-NDPK-B were 18 and 16 kDa, respectively. The autophosphorylated S-NDPK-A and S-NDPK-B displayed isoelectric points (p I) of 5.8 and 6.6, respectively. The shoot NDPKs using NDPs (diphosphonucleosides) as substrates were metal dependent, while these underwent autophosphorylation in the absence of metal. The specificity of S-NDPK-A, S-NDPK-B and root NDPK-B (R-NDPK-B); towards mixtures of purino- and pyrimidino-NDPs were tested by TLC (thin layer chromatography). UDP and CDP (pyrimidino-NDPs) and GDP (purino-NDP) were exclusively phosphorylated, using (γ- 32P) ATP as phosphate donor, by S-NDPK-A and R-NDPK-B, respectively. Both purino- and pyrimidino-NDPs were phosphorylated by S-NDPK-B. The above isoforms also displayed differences in preference towards a mixture of ADP, GDP, dGDP, TDP, dCDP, CDP and UDP in the presence of Cu 2+, Zn 2+, Mg 2+, Mn 2+, Ni 2+, Ca 2+, Hg 2+ or Co 2+. For example, GDP was mainly phosphorylated by R-NDPK-B independently of the metal used, TDP was mainly phosphorylated by S-NDPK-A in the presence of Mg 2+, Mn 2+, Ca 2+ or Co 2+ and S-NDPK-B was capable of phosphorylating more or less independently of the metal used. The purified and characterized NDPK isoforms may play different biological roles according to their preference towards NDPs.

Evaluation of the relative mineral-binding affinities of clinically-relevant bisphosphonates by hydroxyapatite-column chromatography

Evaluation of the relative mineral-binding affinities of clinically-relevant bisphosphonates by hydroxyapatite-column chromatography

Evaluation of the relative mineral-binding affinities of clinically-relevant bisphosphonates by using hydroxyapatite-column chromatography and adsorption isotherms combined with mass spectrometric analysis

Evaluation of the relative mineral-binding affinities of clinically-relevant bisphosphonates by using hydroxyapatite-column chromatography and adsorption isotherms combined with mass spectrometric analysis

Purification, properties and specificity of a NDP kinase from Alyssum murale grown under Ni 2+ toxicity

During the growth of Alyssum murale, a nickel accumulator plant, three root peptides chains of 55, 18 and 16kDa undergo phosphorylation. The intensity of the phosphorylated bands decreased in the course of growth in nutrient solution supplied with 0.5mM Ni(2+). In the shoot only two phosphorylated peptide chains with a size of 18 and 16kDa were detected. These two shoot peptides disappeared on the 19th day of growth in Ni(2+)-exposed plants, while the root peptide of 16kDa continued to be present in less intensity. This peptide was identified as the catalytic subunit of nucleoside diphosphate kinase (NDP kinase: E.C. 2.7.4.6) and was named NDPK-B. The enzyme was purified by means of ammonium sulphate precipitation, DEAE-sepharose and hydroxyapatite column chromatography. NDPK-B was thermostable, displayed a molecular mass of 103,000 and was comprised of six catalytic subunits. The autophosphorylated enzyme displayed an isoelectric point (pI) of 6.5. The NDPK-B autophosphorylation activity was metal-dependent. With regard to the transfer reaction, NDPK-B exhibited the following properties: (a) the enzyme had an optimum pH of 7.6; (b) it was capable of using both (gamma-(32)P) ATP and (gamma-(32)P) GTP as phosphate donors and of using all the available NDPs except dCDP as phosphate acceptors; (c) its activity using NDPs as substrates was metal dependent; (d) in the presence of (gamma-(32)P) GTP as the phosphate donor, it phosphorylated exclusively ADP when a mixture of NDPs was added in the reaction mixture; and, (e) ADP had a very low K(m) value towards 8.4nM. This high affinity towards ADP suggests that the enzyme may play a crucial function in the formation of the amount of ATP necessary for Alyssum murale to survive Ni(2+) stress.

Functional Analysis of Norcoclaurine Synthase in Coptis japonica

(S)-Norcoclaurine is the entry compound in benzylisoquinoline alkaloid biosynthesis and is produced by the condensation of dopamine and 4-hydroxyphenylacetaldehyde (4-HPAA) by norcoclaurine synthase (NCS) (EC 4.2.1.78). Although cDNA of the pathogenesis-related (PR) 10 family, the translation product of which catalyzes NCS reaction, has been isolated from Thalictrum flavum, its detailed enzymological properties have not yet been characterized. We report here that a distinct cDNA isolated from Coptis japonica (CjNCS1) also catalyzed NCS reaction as well as a PR10 homologue of C. japonica (CjPR10A). Both recombinant proteins stereo-specifically produced (S)-norcoclaurine by the condensation of dopamine and 4-HPAA. Because a CjNCS1 cDNA that encoded 352 amino acids showed sequence similarity to 2-oxoglutarate-dependent dioxygenases of plant origin, we characterized the properties of the native enzyme. Sequence analysis indicated that CjNCS1 only contained a Fe(2+)-binding site and lacked the 2-oxoglutarate-binding domain. In fact, NCS reaction of native NCS isolated from cultured C. japonica cells did not depend on 2-oxoglutarate or oxygen, but did require ferrous ion. On the other hand, CjPR10A showed no specific motif. The addition of o-phenanthroline inhibited NCS reaction of both native NCS and recombinant CjNCS1, but not that of CjPR10A. In addition, native NCS and recombinant CjNCS1 accepted phenylacetaldehyde and 3,4-dihydroxyphenylacetaldehyde, as well as 4-HPAA, for condensation with dopamine, whereas recombinant CjPR10A could use 4-hydroxyphenylpyruvate and pyruvate in addition to the above aldehydes. These results suggested that CjNCS1 is the major NCS in C. japonica, whereas native NCS extracted from cultured C. japonica cells was more active and formed a larger complex compared with recombinant CjNCS1.