Hydroxylapatite Column Chromatography Research Articles

Expression and purification of pneumococcal surface protein a of clade 4 in Escherichia coli using hydroxylapatite and ion-exchange column chromatography

Streptococcus pneumoniae is a major pathogen that causes life-threatening diseases, such as pneumonia, otitis media, bacteremia, and meningitis, worldwide and especially in young children and the elderly. Pneumococcal surface protein A (PspA) is a widely studied candidate protein vaccine that represents a promising replacement for current polysaccharide and polysaccharide-conjugate vaccines. In this study, we describe a simple method to produce PspA of clade 4 from an Escherichia coli expression system using hydroxylapatite and ion-exchange chromatography. Using this method, we successfully expressed soluble PspA4 in 10 L of autoinducing culture medium, with a wet-cell yield of 19 g/L and a final PspA4 concentration of 22.8 mg/L. Additionally, we improved PspA4 purity from 17% to 70% in a single step through the use of hydroxylapatite, resulting in acquisition of recombinant PspA4 (>95% purity) at a final yield of 43% from the starting cell-lysis solution. We subsequently verified the secondary structure molecular weight of recombinant PspA4 by circular dichroism and mass spectrometry, respectively. These results demonstrated a highly efficient method for mass producing PspA4 protein and that can also be applied for purification of PspA proteins from other clades.

Purification Protocol of β-mannosyltransferase from pig aorta

β-Mannosyltransferase that transfers mannose to Dol-PP-GlcNAc2 (chitobiose lipid acceptor) from GDP-Man was purified from the microsomes of pig aorta. The purification protocol consisted of the following steps: (i) preparation of microsomal fraction (ii) selective solubilization by Nonident NP-40 nonionic detergent at a protein to detergent ratio of 3:2; (iii) anion exchange chromatography on DE-52 in the presence of 0.4% NP-40, (iv)hydroxylapatite column chromatography, (v) chromatography on HQ anion exchange column, (vi) octyl Sepharose chromatography.

Isolation of two cytochrome P450 forms, CYP2A19 and CYP1A, from pig liver microsomes

Cytochromes P450 comprise a superfamily of proteins that play a crucial role in the biotransformation of numerous chemicals. Purified CYPs can be used e.g. in studies on structure or determining the drug metabolism pathways. In this work, purification of the porcine CYP1A and CYP2A19 to electrophoretic homogeneity from the pig hepatic microsomes using octylamino Sepharose and hydroxylapatite column chromatography is reported. The proteins have been clearly recognized by commercial antibodies against rat and human CYP1A2 (porcine CYP1A) and human CYP2A6 (CYP2A19) respectively, using Western blot. Activities of both enzymes were determined by specific substrates, 7-ethoxyresorufin, 7-methoxyresorufin (CYP1A) and coumarin (CYP2A19). The isolated enzymes show kinetic parameters similar to human counterparts. Taken together, pig cytochromes can be used for the testing of veterinary drug metabolism, useful for the determination of drug residues in meat of pigs. The results obtained show that the pigs may be a suitable model for biotransformation of xenobiotics in humans.

PARTIAL PURIFICATION AND CHARACTERIZATION OF b-GLUCOSIDASE FROM ALOE VERA L. LEAVES

The partial purification of β-glucosidase from the pulp of Aloe vera L. Burm. fil. (sarisabir) leaves and some of its kinetic properties is presented. The fresh leaves of A. vera were used; the gel portion was separated and the remaining leaf pulps were cut into small pieces. The crude extract was prepared by homogenization of the leaf pulps in phosphate buffered saline (PBS), pH 7.4 and subsequent centrifugation. β-Glucosidase active fraction was precipitated by 30%-65% ammonium sulphate from the crude extract. Hydroxylapatite column chromatography resulted in a single peak showing β-glucosidase activity eluted with 200 mM phosphate buffer. The partially purified enzyme showed two protein and a single activity band in polyacrylamide gel electrophoresis. It was found that the enzyme exhibited maximum activity at 50 o C and at pH 4.4. Km and Vmax values for 4-nitrophenyl-β-D-glucopyranoside were 6.8x10 - 4 M and 4,58x10 -3 U, respectively. When β-glucosidase activity was investigated throughout the year, it was found that the activity increased in winter and decreased in summer.

Purification and characterization of lipovitellin from Pacific sauryCololabis saira

Lipovitellin (Lv), the major yolk protein derived from vitellogenin (Vg), was purified from vitellogenic ovaries of Pacific saury Cololabis saira using hydroxylapatite column chromatography followed by gel filtration. The apparent native mass of purified Lv was approximately 420 kDa, while the tertiary structure of Lv revealed by sodium dodecylsulfate—polyacrylamide gel electrophoresis was typical of teleost Lvs, consisting of a heavy chain (∼99 kDa) and a light chain (∼34 kDa). Western blot analysis using rabbit antiserum raised against Pacific saury Lv revealed a specific reaction with a polypeptide (∼194 kDa) that is present in serum from female Pacific saury but not in male serum, suggesting the approximately 194-kDa polypeptide to be the Vg monomer. This study describes the first step toward the development of specific immunoassays for Pacific saury Vg, which will be an effective tool for monitoring the reproductive development of this species.

Carp ( Cyprinus carpio) vitellogenin: purification and development of a simultaneous chemiluminescent immunoassay

Vitellogenin (Vg) was purified from the serum of vitellogenic female carp ( Cyprinus carpio) by hydroxylapatite column chromatography and gel filtration. Vg had an apparent molecular mass of 490 kDa and appeared as two bands corresponding to 190 and 156 kDa after SDS-PAGE under reducing conditions. These bands were immunoreacted in Western blotting using antiserum against carp lipovitellin (anti-Lv) which is an egg yolk protein derived from Vg. The amino acid composition of carp Vg was similar to previous reports of cyprinids. The chemiluminescent immunoassay (CLIA) for carp Vg was developed to quantify serum Vg using purified carp Vg and anti-Lv. Its measurable range was from 1.95 to 1000 ng/ml. The dilution curve in the CLIA of vitellogenic female serum was parallel to the standard curve of purified Vg. The coefficient variations of intra- and inter-assay were less than 5%, respectively. Furthermore, the assay had cross-reactivity with the sera of other female cyprinids (crucian carp and Japanese dace). In fish diets-experiments, Vg was detected in all fish in the fish meal containing soybean (20%) group, but was not detected in almost all of the fish in the fish meal-group. This suggests that a soybean based-diet may induce Vg production in the serum of cultivated carp.

Development of vitellogenin-ELISA, an in vivo bioassay, and identification of two vitellogenesis-inhibiting hormones of the tiger shrimp Penaeus monodon.

A sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification of vitellogenin in the hemolymph of Penaeus monodon is reported. Lipovitellin from the mature ovary was purified using hydroxylapatite column chromatography and used as the standard protein, which was serially diluted from 3336 to 6.51 microg and gave a linear plot. Sensitivity results showed ELISA was insensitive to samples that did not contain vitellogenin or lipovitellin. Specificity results showed the degree of the discrimination of the assay between positive samples (having vitellogenin or lipovitellin) and negative samples (devoid of vitellogenin or lipovitellin). Reproducibility studies showed that the intraassay coefficient of variation was 5.1% (n = 11) and the interassay coefficient of variation was 5.15% (n = 13). Separation of X-organ sinus gland peptides using a reversed phase column gave a total of 37 fractions. Two fractions were found to reduce the hemolymph vitellogenin concentration in a time-dependent manner and could be identified as vitellogenesis-inhibiting hormones I and II.

Purification and characterization of mannose isomerase from Agrobacterium radiobacter M-1.

A mannose isomerase from Agrobacterium radiobacter M-1 (formerly Pseudomonas sp. MI) was purified to electrophoretic homogeneity and characterized. A cell-free extract was separated by ammonium sulfate fractionation, Butyl-Toyopearl 650M, DEAE-Sepharose and hydroxylapatite column chromatography. Its molecular mass was estimated to be 44 kDa by SDS-PAGE and 90 kDa by gel filtration, in which the enzyme is most likely a dimer composed of two identical subunits. The purified enzyme had an optimum pH at 8.0, an optimum temperature at 60 degrees C, a pI of 5.2 and a Km of 20 mM, and specifically converted D-mannose and D-lyxose to ketose. The N-terminal amino acid sequence was identified.

A two-step purification of cytochrome P-450 from adult pig testis by pregnenolone affinity column chromatography.

Adult testicular cytochrome P-450 was purified by a two-step procedure utilizing hydroxylapatite and pregnenolone affinity column chromatography. The cytochrome P-450 was determined to have an isoelectric point of 6.43 on analytical isoelectric focusing. The purified cytochrome P-450 was found to be homogeneous and its molecular weight was estimated to be 52,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectrum with a peak at 448 nm exhibited the absorption spectrum of a typical cytochrome P-450. A purification of 755x was achieved with a yield of 3.09%.

A thermostable diamine oxidase from Vigna radiata seedlings

Diamine oxidase (DAO) (EC. 1.4.3.6.) from the 1-day-old Vigna radiata cv. P105 seedlings grown in the dark was found to be thermotolerant, showing optimal activity at 60°, with an E a of 2.75 kcal/mol estimated at temperatures from 25°–85° from the Arrhenius plot. The enzyme was purified up to 22- fold by ammonium sulphate fractionation, heat denaturation and hydroxylapatite column chromatography. The M r was estimated by gel filtration to be 139 kDa and the SDS–PAGE yielded a single band at M r 68 kDa, indicating the occurrence of two identical subunits in the mungbean DAO. The K m value for putrescine, as the preferred substrate, was 0.15 mM and V max was 0.065 μmol/min. The DAO was inhibited by sulphydryl reagents, copper binding agents, and other mono- and divalent ions.

Purification of cytochrome P-450 from adult pig testis by hydroxylapatite and deoxycorticosterone affinity column chromatography

Adult testicular cytochrome P-450 was purified by a two-step procedure utilizing hydroxylapatite and deoxycorticosterone affinity column chromatography. Cytochrome P-450 was determined to have an isoelectric point of 6.5 on analytical isoelectric focusing. The purified cytochrome P-450 was found to be homogeneous and its molecular mass was estimated to be 52 000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectrum with a peak at 448 nm exhibited the absorption spectrum of a typical cytochrome P-450. A 1000-fold purification was achieved with a yield of 5%.

Purification and Some Properties of Two Enzymes from Rat Liver Cytosol That Catalyze Carbonyl Reduction of 6-tert-Butyl-2,3-epoxy-5-cyclohexene-1,4-dione, a Metabolite of 3-tert-Butyl-4-hydroxyanisole

6-tert-Butyl-2,3-epoxy-5-cyclohexene-1,4-dione (TBE), a metabolite of 3-tert-butyl-4-hydroxyanisole, was converted to 6-tert-butyl-2,3-epoxy-4(R)-hydroxy-5-cyclohexen-1-one ((4R)-TBEH) and 6-tert-butyl-2,3-epoxy-4(S)-hydroxy-5-cyclohexen-1-one ((4S)-TBEH) by TBE-reducing enzymes in rat liver cytosol. Two TBE-reducing enzymes (TBE-R1 and TBE-R2) were purified 18- and 117-fold, respectively, to apparent homogeneity from rat liver cytosol using DEAE-Sephacel, Blue Sepharose CL-6B, hydroxylapatite, and Sephadex G-100 column chromatography. Gel filtration and sodium dodecyl sulfate–polyacrylamide gel electrophoresis indicated that both enzymes were monomeric. The purified TBE-R1 and TBE-R2 had molecular weights of 37 and 35 kDa and isoelectric points of 6.5 and 5.8, respectively. Both enzymes had an optimum pH of about 5.5 with TBE as substrate. TBE-R1 utilized NADH or NADPH equally as cofactor, and theKmvalues of NADH and NADPH for TBE with TBE-R1 were estimated to be 15 and 29 μM, respectively. On the other hand, TBE-R2 specifically utilized NADPH and theKmvalue for TBE was estimated to be 92 μM in the presence of NADPH. Both enzymes reduced aromatic aldehydes, ketones, and quinones at higher rates. In addition, TBE-R2 reduced and oxidized 3-ketosteroids at a higher rate in the presence of NAD(H) and/or NADP(H). Both enzyme activities were inhibited by quercitrin orp-chloromercuribenzoic acid, but little inhibition was observed with phenobarbital or pyrazole. Dicoumarol inhibited significantly TBE-R1 activity but not TBE-R2 activity. In the conversion of TBE to TBEH, TBE-R1 preferentially reduced TBE to (4R)-TBEH, whereas TBE-R2 preferred the reduction of TBE to (4S)-TBEH.

Purification, cDNA Cloning, and Expression of UDP-Gal: Glucosylceramide β-1,4-Galactosyltransferase from Rat Brain

Lactosylceramide synthase is an enzyme that catalyzes the transfer of galactose from UDP-Gal to glucosylceramide, and thus participates in the biosynthesis of most glycosphingolipids in mammals. We purified this enzyme over 61,000-fold to near homogeneity with a 29. 7% yield from rat brain membrane fractions. The isolation procedure included solubilization with Triton X-100, affinity chromatography on wheat germ agglutinin-agarose and UDP-hexanolamine-agarose, and hydroxylapatite column chromatography, followed by ion exchange chromatography. The final preparation migrated as a broad band with an apparent molecular mass of 61 kDa on SDS-polyacrylamide gel electrophoresis. This apparent molecular mass was reduced to 51 kDa by N-glycanase digestion, suggesting that the enzyme has a glycoprotein nature. The enzyme required Mn2+ for its activity, and glucosylceramide was its preferred substrate. The cDNA for the enzyme was cloned from a rat brain cDNA library. The cDNA insert encoded a polypeptide of 382 amino acid residues, with a molecular weight of 44,776. The polypeptide contained eight putative glycosylation sites and a 20-amino acid residue transmembrane domain at its N terminus. Amino acid sequence homology analysis revealed that this enzyme shared 39% homology with mouse beta-1, 4-galactosyltransferase (EC 2.4.1.38), which catalyzes the transfer of Gal to beta-1,4-GlcNAc in glycoproteins.

Purification and characterization of a lipase from Pseudomonas aeruginosa KKA-5 and its role in castor oil hydrolysis

An extracellular lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) from Pseudomonas aeruginosa KKA-5 hydrolyzed castor oil by 90%. Purification of this castor oil-hydrolyzing lipase included ammonium sulfate precipitation and successive hydroxylapatite column chromatography. The enzyme was purified 518-fold. It was homogeneous electrophoretically and its molecular weight was estimated to be 30 kDa. The enzyme was stable up to 45°C and retained its activity in the alkaline pH range. Lipase was highly stable in the presence of aqueous organic solvents like methanol and ethanol. It was weakly inhibited in the presence of acetone. The anionic surfactant, sodium dodecyl sulfate, was inhibitory while the cationic surfactants, Triton X-100 and Tween-80 appreciably enhanced activity. Lipase was stabilized significantly by Ca2+. Inactivation of the enzyme by EDTA was overcome by sequential CaCl2 treatment. This finding suggests the existence of a calcium-binding site in Pseudomonas aeruginosa KKA-5 lipase.

Separation of three P450 isozymes from liver microsomes of gilthead seabream treated with ß‐NF and partial purification of cytochrome P4501A1

Three distinct cytochrome P450 isozymes, P4501A1, P4502B and an unidentified P450 isozyme were separated and isolated from beta-NF-treated gilthead seabream liver microsomes. Cytochrome P4501A1 was partially purified from beta-NF-treated gilthead seabream liver microsomes in the presence of detergents Emulgen 913 and cholate and protease inhibitors using two DEAE-cellulose, Porapak Q and two hydroxylapatite column chromatographies. The overall yield of purified P4501A1 was 1.2% with respect to microsomal total P450 with a specific content of 3 nmol/mg protein. The purified P4501A1 was characterized with respect to spectral, electrophoretic, biocatalytic and immunochemical properties, which are found to be similar to P4501A1s purified from other teleost species such as trout, scup, cod and perch suggesting that the P450 we have purified belongs to CYP1A1.

Evidence of two catalytically active carnitine medium/long chain acyltransferases in rat liver peroxisomes

Peroxisomal matrix proteins were extracted from the highly purified peroxisomes with sodium pyrophosphate, and the membranes were sedimented by high speed centrifugation. Biochemical marker enzyme analyses revealed a quantitative release of a number of well-known peroxisomal matrix proteins from the purified peroxisomes. In contrast, carnitine medium/long chain acyltransferase activity, assayed with decanoyl-CoA and palmitoyl-CoA as substrates, was equally distributed in the membrane and the matrix fractions. The matrix and the membrane enzyme activities were differentially affected by a number of detergents. The enzyme in the membrane fraction showed higher malonyl-CoA sensitivity compared to the enzyme in the matrix fraction. The enzyme(s) from the purified peroxisomes or the peroxisomal membranes was quantitatively solubilized by sodium cholate, and the cholate-solubilized enzyme retained malonyl-CoA sensitivity. The membrane enzyme was separated from the matrix enzyme by hydroxylapatite column chromatography. The separation of the membrane enzyme or the matrix enzyme by hydroxylapatite column chromatography resulted in loss of malonyl-CoA sensitivity. The partially purified membrane and the matrix enzymes showed broad substrate specificity, and the highest enzyme activities for both were observed with decanoyl-CoA. In contrast to the matrix enzyme, the membrane enzyme was strongly inhibited by high concentrations (> or = 50 microM) of acyl-CoAs (> 10 carbons in length). The matrix enzyme showed a 2.5-fold lower Km for carnitine compared to the membrane enzyme. The catalytic properties of the partially purified matrix enzyme appear to be similar to the well-characterized peroxisomal carnitine octanoyltransferase, though we find highest activity with decanoyl-CoA rather than octanoyl-CoA as a substrate. The data presented clearly indicate that the membrane and the matrix enzyme activities are due to different proteins.

Isolation and Partial Characterization of the NADH Dehydrogenase Complex from Barley Chloroplast Thylakoids

Using chloroplasts from barley leaves we attempt to purify and partially characterize the NADH dehydrogenase complex. The enzymatic activity was assayed as NADH-ferricyanide and NADH-nitro blue tetrazolium oxidoreductase. Analyzed by SDS-polyacrylamide gel electrophoresis and subsequent enzymatic renaturation, the chloroplast soluble fraction contains principally a 66 kDa enzyme. The membranous fraction solubilized with deoxycholate and analyzed by native electrophoresis and NADH-nitro blue tetrazolium staining revealed three enzymes: one with similar electrophoretic mobility to that described for the soluble enzyme, another one which is a complex separated in 3% polyacrylamide gel and a third one, another complex separated in the top of the 5-22% linear polyacrylamide gel gradient. The complex polypeptidic patterns were similar but different to those found for any thylakoidal proteinic complex known. Nine major polypeptides were detected in the complex polypeptidic patterns, four of them constituents of the small size thylakoid enzyme. The molecular masses of six polypeptides agreed with those indicated as encoded by 6 chloroplast ndh genes. All the enzymes, including the 66 kDa soluble enzyme, contained a S3 kDa polypeptide, which is probably the NADH-binding complex subunit. Isoelectric focusing of the thylakoidal enzyme points to a basic isoelectric point. Ion-exchange or hydroxylapatite column chromatography followed by native electrophoresis of the active fractions only separated the small size enzyme, which showed complex inactivation.

Purification of a Lysophospholipase from Bovine Brain That Selectively Deacylates Arachidonoyl-substituted Lysophosphatidylcholine

A high activity lysophospholipase A (lysoPLA) was purified from the soluble fraction of bovine brain. The separation included sequential DEAE-Sephacel, phenyl-Sepharose FF, heparin-Sepharose CL-6B, and Q-Sepharose FF column chromatography. Mono Q, Sephacryl S300HR, and hydroxylapatite column chromatography in the presence of the detergent CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate) and glycerol further purified the activity to 17,000-fold. The enzyme was purified to homogeneity by polyacrylamide gel electrophoresis using nondenaturing conditions. The pure enzyme migrated as a single polypeptide of 95 kDa mass by SDS-polyacrylamide gel electrophoresis and deacylated arachidonoyl-lysophosphatidylcholine (ara-lysoPC) at rate of 70 micromol/(min mg). The enzyme showed selectivity for arachidonoyl-substituted lysoPC, since palmitoyl-lysoPC was deacylated at a much lower rate (7 micromol/(min mg)). LysoPLA activity was maximal at pH 7.4-8.0 and was increased 1.3-fold by MgCl2 (5 mM). By including MgCl2, however, the range of optimal activity was expanded to pH values up to 9.0. The 95-kDa protein also deacylated arachidonoyl groups from 1-O-hexadecyl-2-arachidonoyl-PC (PLA2 activity) at a rate of 15 micromol/(min mg). Moreover, the deacylation of arachidonoyl groups from diacylPC was greatly increased by including purified bovine brain PLA1 in the reaction mixture. Thus, the same 95-kDa polypeptide catalyzed both lysoPLA and PLA2 activities, but the rate of arachidonoyl group deacylation was increased by prior sn-1 deacylation. Finally, the 95-kDa polypeptide cross-reacted with antibodies raised against a human recombinant cPLA2, implying that the 95-kDa protein is structurally similar to cPLA2. Additionally, these data suggest that the combined actions of PLA1 and the 95-kDa protein generate significant amounts of free arachidonic acid in the brain.

Photoinhibition of Photosystem I electron transfer activity in isolated Photosystem I preparations with different chlorophyll contents

Photoinhibition of the light-induced Photosystem I (PS I) electron transfer activity from the reduced dichlorophenol indophenol to methyl viologen was studied. PS I preparations with Chl/P700 ratios of about 180 (PS I-180), 100 (PS I-100) and 40 (PS I(HA)-40) were isolated from spinach thylakoid membranes by the treatments with Triton X-100, followed by sucrose density gradient centrifugation and hydroxylapatite column chromatography. White light irradiation (1.1 × 10(4)μE m(-2) s(-1)) of PS I-180 for 2 hours bleached 50% of the chlorophyll and caused a 58% decrease in the electron transfer activity with virtually no loss of the primary donor, P700. The flash-induced absorbance change showed the decay phase with a half time of about 10 μs that was attributed to the P700 triplet, suggesting that the photoinhibitory light treatment caused the destruction of the PS I acceptor(s), Fx and possibly A1. PS I-100 was similarly photobleached by the irradiation and the electron transfer activity decreased. There was, however, no apparent photoinhibition of the electron transport activity in PS I(HA)-40. Photoinhibition similar to that seen in PS I-180 also occurred in membrane fragments that were isolated without any detergent from a PS II-deficient mutant strain of the cyanobacterium Synechocystis sp. PCC 6803. PS I-180 was not photoinhibited under anaerobic conditions. The production of superoxide and fatty acid hydroperoxide during white light irradiation was significantly greater in PS I-180 than in PS I(HA)-40. The mechanism of photoinhibition in PS I preparations is discussed in relation to the formation of toxic oxygen molecules.

The Role of Pea Chloroplast [alpha]-Glucosidase in Transitory Starch Degradation.

Pea chloroplastic [alpha]-glucosidase (EC 3.2.1.20) involved in transitory starch degradation was purified to apparent homogeneity by ion exchange, reactive dye, hydroxylapatite, hydrophobic interaction, and gel filtration column chromatography. The native molecular mass and the subunit molecular mass were about 49.1 and 24.4 kD, respectively, suggesting that the enzyme is a homodimer. The enzyme had a Km of 7.18 mM for maltose. The enzyme's maximal activity at pH 7.0 and stability at pH 6.5 are compatible with the diurnal oscillations of the chloroplastic stromal pH and transitory starch accumulation. This pH modulation of the [alpha]-glucosidase's activity and stability is the only mechanism known to regulate starch degradative enzymes in leaves. Although the enzyme was specific for the [alpha]-D-glucose in the nonreducing end as the glycon, the aglycon moieties could be composed of a variety of groups. However, the hydrolysis rate was greatly influenced by the aglycon residues. Also, the enzyme could hydrolyze glucans in which carbon 1 of the glycon was linked to different carbon positions of the penultimate glucose residue. The ability of the [alpha]-glucosidase to hydrolyze [alpha]-1,2- and [alpha]-1,3-glucosidic bonds may be vital if these bonds exist in starch granules because they would be barriers to other starch degradative enzymes. This purified pea chloroplastic [alpha]-glucosidase was demonstrated to initiate attacks on native transitory chloroplastic starch granules.