Properties of androsterone-sulfating sulfotransferase in female rat liver

Some properties of androsterone (AD)-sulfating sulfotransferase (ST) present in female rat livers were characterized. Based on the substrate specificities of the enzyme preparation obtained by anion exchange chromatography and 3'-phosphoadenosine 5'-phosphate (PAP)-agarose affinity chromatography, AD-ST was supposed to be among isoenzymes of hydroxysteroid STs. The identity of the AD-ST with the isoenzymes of hydroxysteroid ST, however, remains unclear at present. The enzyme preparation revealed a wide range of native molecular weight with a major Mr of some 600000. The AD-ST did not appear to have a homogeneous isoelectric point, because the enzymatic activity was spread over a wide range of the pH gradient, centering around pH 6.6 on chromatofocusing. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the AD-ST showed a subunit with Mr of 30000, which was similar to the hydroxysteroid STs purified previously. Under denaturing conditions the subunit was demonstrated to be composed of three protein species containing distinct pI values (pI 6.1, 6.7 and 7.2). The AD-ST was thus supposed to be an oligomer with high molecular weight, in which the subunits of different pI values are assembled in various association numbers.     

Biological Characterization of a Pituitary Monkey Lutropin Preparation after Chromatofocusing or after High Performance Anion Exchange Chromatography

Abstract

The Pharmacia Fast Protein Liquid Chromatography System equiped with a Mono P HR 5/10 chromatofocusing column was used to characterize the distribution of different bioactive forms of a cynomolgus (Macaca bascicularis) pituitary lutropin preparation. The results were compared with the profiles obtained after running the preparation on a Waters DEAE anion exchange column using eluants in the pH range from 6.0 to 8.0. The distribution of preserved biological activity were studied in respective eluted fractions from the different experiments of the applied preparation. Seventy-six percent of the bioactivity was recovered after chromatofocusing whereas preserved bioactivity after ion exchange chromatography ranged between 61–78% at pH 6.5 to 7.5. No bioactivity was restored after elution at pH 6.0 or 8.0.     

Identification of estrogenic/anti-estrogenic compounds in diesel exhaust particulate extract

Diesel exhaust particulate extract (DEPE) was obtained from diesel exhaust particulates with Soxhlet extraction using dichloromethane. After separating DEPE into 11 fractions by liquid-liquid extraction, the neutral fraction (N) showed anti-estrogenic activity and the weak acid (phenol) fraction (WA(P)) showed estrogenic and anti-estrogenic activities by a yeast two-hybrid assay system expressing human estrogen receptor alpha. Both fractions were thoroughly fractionated by silica gel column chromatography and reversed-phase HPLC. In the WA(P) fraction, 3-methyl-4-nitrophenol and 2,6-dimethyl-4-nitrophenol were identified by LC-MS/MS as estrogenic compounds. This is the first study to identify 2,6-dimethyl-4-nitrophenol in DEPE and the first study to show that it is an estrogenic compound. In the N fraction, 1-hydroxypyrene was also identified by LC-MS/MS as an anti-estrogenic compound.     

Interaction between indigo and adsorbed protein as a major factor causing backstaining during cellulase treatment of cotton fabrics

A model microassay system was developed to measure indigo backstaining on cotton fabrics in the presence of enzymes on a small laboratory scale. Backstaining indexes for 11 cellulase samples were measured, and the enzymes were ranked from lower to higher backstaining. Two multienzyme cellulase preparations were separated into fractions using chromatofocusing on a Mono P column. Adsorption ability and backstaining properties of purified enzyme fractions were studied. Evidence was obtained that protein adsorption on cotton fabrics is a crucial parameter causing backstaining (both for crude cellulase samples and purified enzyme components).     

柱状假丝酵母脂肪酶同工酶的一种高效液相色谱分离新方法

建立一种“疏水界面亲和色谱”分离柱状假丝酵母脂肪酶同工酶的高效液相色谱新方法。将商品化的CRL经离子交换色谱分离为两个同工酶组分 (CRLA和CRLB) ,在极低离子强度下 ,根据同工酶活性中心周围处于“开放”构象的疏水腔具亲疏水界面的特性 ,用疏水界面亲和色谱在NucleosilC4 (10 μ ,3 0 0 ,2 5 0× 4.60mm)柱上将CRLA和CRLB都分离为 4种同工酶组分。疏水界面亲和色谱非常适用于分离这种结构差异轻微的同工酶组分     

Isolation and partial characterization of angiotensinase A and aminopeptidase M from urine and human kidney by lectin affinity chromatography and high-performance liquid chromatography

Angiotensinase A (ATA) and aminopeptidase M (APM) were partially purified from human urine specimens and human kidney particles using wheat germ lectin affinity chromatography, anion-exchange Fast Protein Liquid Chromatography (FPLC) (Mono Q), chromatofocusing (Mono P, FPLC) and Superose 12 gel filtration. APM, a globular 5-nm glycoprotein, is localized in the brush border membrane of the proximal tubule; angiotensin II-degrading ATA is present on glomerular endothelia and podocytes and, to lesser extent, in the brush border. For the first time, both peaks of ATA and APM activity from urine samples were separated by the above-mentioned techniques with only slight overlap; ATP (146,000 dalton: pI4.8) was enriched more than 20-fold and APM (153,000 dalton, pI4.7) more than 50-fold compared with the activity of the starting material. Using similar separation steps, ATA and APM solubilized from kidney particles could not be resolved into two distinct peak fractions, however, except after hydrophobic interaction chromatography. Thus urine is a major source for the preparation of individual ATA and APM fractions, necessary to generate specific anti-enzyme antibodies for diagnostic purposes.     

Antioxidant activity of lignin phenolic compounds extracted from kraft and sulphite black liquors

The antioxidant activity of the phenolic compounds present in industrial black liquors obtained from the two cooking processes (kraft and sulphite) used in Portugal to produce Eucalyptus globulus pulp was evaluated. The black liquors treated at several pH values were extracted with ethyl acetate. Phenolic fractions were further separated by liquid chromatography of the crude extracts of kraft liquor at pH = 6 and sulphite liquor at the original pH. Total phenolic content was determined in terms of gallic acid equivalents (Folin-Ciocalteu colorimetric method), and the antioxidant activity in the crude extracts at several pH values and in the separated fractions was measured using the DPPH test for radical scavenging capacity. The total phenolic content of crude extracts and separated fractions ranged from 92.7 to 181.6 and from 91.6 to 1,099.6 mg GAE/g, respectively, while the antioxidant activity index (AAI) ranged from 2.20 to 3.41 and from 2.21 to 11.47 respectively, showing very strong antioxidant activity in all studied cases. The fractions separated by column chromatography were submitted to mass spectrometry analysis and the results were compared to others in the literature of natural products, mainly from Eucalyptus, and the characteristic bands of functional groups were identified by 1H-NMR and FTIR. These methods allowed the identification of 17 phenolic compounds.     

Chromatofocusing of sialoglycoproteins

Sialoglycoproteins of different sialic acid contents have been separated from each other by chromatofocusing on the ion exchanger PBE 94 using gradients of pH 4.00 down to pH 1.00. The technique is much faster than isoelectric focusing, apparently does not result in desialylation of the sialoglycoproteins and can handle with ease 10 ng to 50 mg quantities of protein on a 22 X 0.9 cm column. The technique revealed that commercial preparations of fetuin and human acid glycoprotein contained several components. Glycophorin, desialylated by controlled neuraminidase treatment, was fractionated by chromatofocusing into several components which differed in sialic acid content and in ability to inhibit haemagglutination by wheat germ agglutinin and encephalomyocarditis and influenza viruses.     

高效液相色谱法测定环境水中痕量4,4′-二氨基联苯、4-硝基酚和苯酚

采用ＰｈｅｎｏｍｅｎｅｘＳｐｈｅｒｅｘＣ１８色谱柱,以含５０ｍｍｏｌ／Ｌ乙酸－乙酸钠缓冲溶液（ｐＨ６．０）的Ｖ（乙腈）∶Ｖ（乙醚）∶Ｖ（水）＝１２∶１０∶７８混合溶液为流动相,用分光光度法检测,检测波长为２７５ｎｍ,在１６ｍｉｎ内实现了４,４′－二氨基联苯、４－硝基酚和苯酚的同时分离测定。检测限分别为０．１４,０．１９和０．０８ｎｇ。标准回收率分别为９８．８５％,９８．１５％和９８．１０％。方法灵敏度高,用于环境水样分析时结果令人满意。     

High-performance cation-exchange chromatofocusing of proteins

Chromatofocusing using high-performance cation-exchange column packings, as opposed to the more commonly used anion-exchange column packings, is investigated with regard to the performance achieved and the range of applications possible. Linear or convex gradients in the range from pH 2.6 to 9 were formed using a variety of commercially available column packings that provide a buffering capacity in different pH ranges, and either polyampholytes or simple mixtures having a small number (three or fewer) of buffering species as the elution buffer. The resolutions achieved using cation-exchange or anion-exchange chromatofocusing were in general comparable, although for certain pairs of proteins better resolution could be achieved using one type of packing as compared to the other, evidently due to the way electrostatic charges are distributed on the protein surface. Several chromatofocusing methods were investigated that take advantage of the acid-base properties of commercially available cation-exchange column packings. These include the use of gradients with a composite shape, the use of very low pH ranges, and the use of elution buffers containing a single buffering species. The advantages of chromatofocusing over ion-exchange chromatography using a salt gradient at constant pH were illustrated by employing the former method and a cation-exchange column packing to separate beta-lactoglobulins A and B, which is a separation reported to be impossible using the latter method and a cation-exchange column packing. Trends in the apparent isoelectric points determined using cation- and anion-exchange chromatofocusing were interpreted using applicable theories. Results of this study indicate that cation-exchange chromatofocusing is a useful technique which is complementary to anion-exchange chromatofocusing and isoelectric focusing for separating proteins at both the analytical and preparative scales.     

Proteomic analysis of rat plasma by two-dimensional liquid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry

The proteomic analysis of plasma and serum samples represents a formidable challenge due to the presence of a few highly abundant proteins such as albumin and immunoglobulins. Detection of low abundance protein biomarkers therefore requires either the specific depletion of high abundance proteins using immunoaffinity columns and/or optimized protein fractionation methods based on charge, size or hydrophobicity. Here we describe a two-dimensional (2D) liquid chromatography separation method for the fractionation of rat plasma. In the first dimension proteins were separated by chromatofocusing according to their isoelectric point (pI). In the second dimension, proteins were further fractionated by non-porous, reversed-phase chromatography according to their hydrophobicity. The data from both separations was displayed as a 2D protein expression map of pI versus retention time (relative hydrophobicity). Both separations were carried out on the ProteomeLab PF 2D system (Beckman Coulter), an instrument platform that provides a high degree of automation and real-time monitoring of the separation process. The reproducibility of the first-dimension separation was evaluated in terms of pH gradient formation. The second-dimension separation was evaluated in terms of peak retention times on the reversed-phase column. We found in four consecutive chromatofocusing separations that the pH gradient differed by less than 0.2 pH units at any time during the elution step. Second dimension retention times of peaks from identical pI fractions differed by less than 7 s in six consecutive separations. Each 2D separation generated a total of 540 fractions which were analyzed by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). We detected approximately 275 peptides and proteins with molecular masses ranging from 3 to 225 kDa. Most fractions were found to contain multiple low and high molecular weight proteins. Differential display of 2D protein expression maps from retinol-sufficient and -deficient rat plasma samples identified a fraction with several proteins that appeared to be down-regulated in the vitamin A-deficient animal. Quantitative proteomic analysis of complex samples such as plasma is still a difficult task. We discuss the potential of this approach for biomarker discovery and address the experimental challenges that remain.     

超高效液相色谱-串联质谱法测定染发剂中7种酚类化合物

提出了应用超高效液相色谱-串联质谱法同时测定染发剂中4-氨基-2-硝基苯酚、3-二乙氨基酚、2-氨基-4-氯苯酚、2-氨基-5-硝基苯酚、2-氨基-3-硝基苯酚、1,7-二羟基萘酚和2,3-二羟基萘酚等7种酚类化合物的方法。采用甲醇萃取染发剂中酚类成分,经WatersAcquityUPLCTMBEHC18色谱柱分离,外标法定量,多反应监测模式采集质谱数据。7种酚类化合物的检出限(3S/N)均低于50.0μg.L-1。在10,20,50μg.g-1三个添加水平下,7种酚类化合物的回收率在68.8%～112.5%之间,相对标准偏差(n=6)在1.58%～12.61%之间。     

Possibilities for speciation of Al-citrate and other negatively charged Al complexes by anion-exchange FPLC-ICP-AES

An anion-exchange fast protein liquid chromatographic-inductively coupled plasma atomic emission spectrometric procedure (FPLC-ICP-AES) was developed for speciation of Al-citrate and other negatively charged Al complexes. FPLC separations were carried out on a Mono Q HR 5/5 strong anion-exchange FPLC column over a pH range from 3.5 to 11.0. An aqueous-NaNO(3) (4 mol dm(-3)) linear gradient elution was applied over 10 min for separation of a particular Al species. The separated Al species were determined in 0.5 cm(3) eluate fractions ;off line' by ICP-AES. Under optimal analytical procedures Al-citrate was separated from Al-oxalate and Al-EDTA in a neutral pH range. Good reproducibility of the FPLC-ICP-AES procedure was obtained for determination of a particular Al species at optimal measurement conditions (RSD +/-2%). Al(3+) and neutral Al-citrate species were strongly adsorbed on the column resin and did not interfere with the separation of negatively charged Al complexes. Al(OH)(4)(-) species were separated from Al-citrate in an alkaline pH region, but quantitatively determined only at a pH of 11.0. The distribution of Al species over a pH range from 3.5 to 11.0 agreed with the reported calculated data. The limit of detection (3sigma basis) for separated Al species was 0.1 mug cm(-3).     

High-performance chromatofocusing using linear and concave pH gradients formed with simple buffer mixtures. II. Separation of proteins

The separation of proteins using high-performance chromatofocusing with linear or concave pH gradients formed using simple mixtures of buffering species in the elution buffer is investigated experimentally. The separation achieved is comparable to that using polyampholyte elution buffers with these types of systems. More specifically, protein band widths at one half of the band height in the range between 0.1 and 0.025 pH units were observed, and good resolution was achieved of protein variants differing by a single amino acid residue in separation times of 30 min or less. An especially useful elution buffer is investigated that contains only four buffering species and that produces a linear pH gradient in the range between pH 9.5 and 6.0 when used together with a particular high-performance column packing made specifically for chromatofocusing. This elution buffer and column packing combination is evaluated by using it for the chromatofocusing of equine myoglobin and human hemoglobin variants. Additional applications are described in which a polyethyleneimine derivatized silica column packing and a pH gradient that is concave in shape are used for the separation of proteins in an E. coli cell lysate.     

Two-step purification of cytochrome P-450 from rat liver microsomes using high-performance liquid chromatography

Cytochrome P-450 from rat liver microsomes treated with phenobarbital (PB) was separated into six fractions, as was cytochrome P-450 treated with 3-methylcholanthrene (MC), by high-performance liquid chromatography (HPLC) with an anion-exchange column. PB and MC induced three forms and one form of cytochrome P-450, respectively. The major forms induced by PB and by MC were further purified to apparent homogeneity based on sodium dodecyl(lauryl)sulphate--polyacrylamide gel electrophoresis by HPLC using a hydroxyapatite column. These new HPLC techniques are simple, rapid and useful for the purification of major forms of cytochrome P-450 from solubilized microsomes.     

Effect of buffer concentration on gradient chromatofocusing performance separating protiens on a high-performance DEAE column

Gradient chromatofocusing is a recently developed chromatographic technique that overcomes the limitations of conventional chromatofocusing. This technique employs a HPLC gradient system and simple low-molecular-mass buffer components to generate linear or other function pH gradients on ion-exchange columns. Results of the present work show a superior separation of beta-lactoglobulin A and B in gradient chromatofocusing compared to salt gradient chromatography using the same DEAE column, with an optimized resolution of 2.3 obtained with gradient chromatofocusing compared to 1.1 obtained with NaCl gradients at constant pH. A significant advantage of the gradient chromatofocusing technique over the conventional chromatofocusing technique is its ability to employ a relatively wide range of buffer concentrations in the mobile phase, the effect of which is studied in the present work. Five proteins (conalbumin, ovalbumin, bovine serum albumin, beta-lactoglobulin A and B) are chromatographed on a DEAE-polymethacrylate HPLC anion-exchange column using the same approximately linear pH gradient profile but different mobile phase buffer concentrations. Results show a significant effect of buffer concentration on peak width, separation factor and resolution. For example, resolution increases from 1.5 to 2.3 in the separation of beta-lactoglobulin A and B when the concentration of each of the components in the 100% elution buffer is increased from 6.25 to 25.0 mM (with the same outlet pH gradient). This separation trend is also seen in the chromatography of ovalbumin from a commercial source, noting a progressive increase in resolution of two peaks in the sample (resolution increased from 0.7 to 2.4) when the concentration of each of the components in the 100% elution buffer is increased from 6.25 to 37.5 mM (same outlet pH gradient). The gains in the resolution are attributed to an increase in the separation factor, since the peak widths are generally noted to also increase with increased buffer concentration. These results point to a significant interplay between buffer concentration and pH, which is not effectively exploited in either conventional chromatofocusing or in conventional ion-exchange chromatographic procedures employing salt gradient elution at constant pH. Gradient chromatofocusing has the ability of optimizing both parameters, thus providing it with unique capabilities in protein separations.     

Extracellular oxidases purified fromCoriolus versicolor

Studies on the extracellular enzymes ofCoriolus versicolor have resulted in the isolation and purification of several proteins that have the potential to act as redox enzymes.C. versicolor was cultured on a glucose-amino acid medium in a large-scale fermenter (60 L) with 2,5-xylidine added to induce the production of extracellular laccase. Proteins were precipitated from the growth medium with ammonium sulfate, and separated by ion-exchange chromatography on DEAE-Sephadex. Further separation of glycoproteins was achieved by affinity chromatography on Concanavalin-A-Sepharose. Polyacrylamide gel electrophoresis on SDS (sodium dodecyl sulfate) and LDS (lithium dodecyl sulfate) gels, isoelectric focusing, and chromatofocusing have been used to establish purity of the proteins and their isoelectric points. Laccase B has been isolated and separated into five fractions by chromatofocusing, with isoelectric points of the fractions varying between pH 4.5 and 6.5. The relative specificity of these fractions towards monophenolic and diphenolic substrates has been investigated. Laccase A was found to differ from laccase B in showing only two bands on isoelectric focusing, with isoelectric points between pH 3.0 and 3.5. Two other proteins isolated from the growth medium were both hemecontaining proteins with interesting spectral properties. One was a “peroxidasetype” heme that could bind carbon monoxide to the iron in the heme, suggesting that the heme may bind oxygen and so function as an oxidase. It reacted with hydrogen peroxide to liberate hydroxyl radicals, but this reaction with hydrogen peroxide resulted in the destruction of the heme center. The real role of this protein is unclear, but several possibilities will be investigated. The second heme-containing protein isolated had different spectral properties from the “peroxidase-type” heme previously described. It had spectral characteristics of a b-type cytochrome in association with a flavin prosthetic group. It appeared to have some similarities to cellobiose oxidase, a heme flavoprotein previously isolated fromSporotrichum pulverulentum, although its molecular weight was 50,100 daltons compared with the 93,000 reported for cellobiose oxidase. Further characterization of this protein will be described.     

Quantification of testosterone and epitestosterone in biological samples by capillary electrophoresis with immunoaffinity extraction

Testosterone is one of the most common doping drugs abused by athletes. Therefore, it is necessary to develop a sensitive and simple method to monitor testosterone and its epimer epitestosterone. An off-line immunoaffinity extraction followed by capillary electrophoresis for simultaneous determination of testosterone and epitestosterone has been described in this paper. Anti-epitestosterone monoclonal antibody which is specific to both testosterone and epitestosterone had been prepared and immobilized on a Sepharose 4B stationary phase. The immunoaffinity column was used for sample cleanup, extraction and preconcentration. After elution and reconstitution, testosterone and epitestosterone in the sample were separated and quantified by micellar electrokinetic chromatography(MEKC) using the borate buffer (200 mM borate, pH 8.7) containing 40 mM sodium cholate as a chiral selector. The immunoaffinity column was evaluated in different parameters such as the retention mechanism, selectivity, binding capacity, elution protocol, and reusability. The separation of these two compounds by MEKC was also optimized. Limit of detection for testosterone and epitestosterone were 5 and 23 ng mL⁻¹, respectively. It was satisfactory to apply this method to analyze testosterone and epitestosterone in spiked urine sample with the recoveries from 78% to 109%.     

Potential for the speciation of Zn using fast protein liquid chromatography (FPLC) and convective interaction media (CIM) fast monolithic chromatography with FAAS and electrospray (ES)-MS-MS detection

Analytical procedures were developed for the speciation of Zn using fast protein liquid chromatography (FPLC), flame atomic absorption spectrometry (FAAS) and convective interaction media (CIM) fast monolithic chromatography with FAAS and electrospray (ES)-MS-MS detection. The investigation was performed on synthetic solutions (2 microg cm-3 Zn) of hydrated Zn2+ species and Zn complexes with citrate, oxalate and EDTA (ligand-to-Zn molar ratio 100:1) over a pH range from 5.4 to 7.4. It was found that Zn interacts with various buffers and the careful adjustment of the pH with diluted solutions of KOH is, therefore, required. FPLC separations were carried out on a Mono Q HR 5/5 strong anion-exchange column, applying an aqueous 1 mol dm(-3) NH4NO3 linear gradient elution over 15 min, at a flow rate of 1.0 cm3 min(-1). The separated Zn species were determined in 1.0 cm3 eluate fractions "off line" by FAAS. Speciation of Zn was also performed on a weak anion-exchange CIM DEAE fast monolithic disc by applying an aqueous 0.4 mol dm(-3) NH4NO3 linear gradient elution over 7.5 min, at a flow rate of 2.0 cm3 min(-1) and determination of the separated Zn species in 1.0 cm3 eluate fractions "off line" by FAAS. Zn-binding ligands in separated fractions were also characterized by electrospray (ES)-MS-MS analysis. The CIM DEAE disc was found to be more efficient in the separation of negatively charged Zn complexes than the Mono Q FPLC column. On the CIM DEAE disc Zn-citrate was separated from both Zn-oxalate and from Zn-EDTA. All these species were also separated from hydrated Zn2+, which was eluted with the solvent front. This method has an advantage over commonly used analytical techniques for the speciation of Zn which are only able to distinguish between labile and strong Zn complexes. Good repeatability of the measurements (RSD 2-4%), tested for six parallel determinations (2 microg cm(-3) Zn) of Zn-EDTA, Zn-citrate and Zn-oxalate was found at a pH of 6.4 on a CIM DAEA disc. The limit of detection (3s) for the separated Zn species was 10 ng cm(-3). The proposed analytical procedure was applied to the speciation of Zn in aqueous soil extracts and industrial waste water from a lead and zinc mining area.     

Separation of pectin methylesterase isoenzymes from tomato fruits using short monolithic columns

One of the main forms of tomato pectin methylesterase (PME; EC 3.1.1.1.1) that is applicable to the food industry was isolated from fresh tomato fruit. The extraction of the PME isoenzymes involved washing the fresh tomato flesh with water in order to remove sugars and than solubilizing the enzymes with a diluted HCl solution at pH 1.6. The extract was then neutralized to pH 7.4 using buffer solution. After filtration, the solution was directly fractioned using Convective Interaction Media (CIM) short monolithic disk column bearing sulfonyl (SO3) groups and using a linear gradient from 0 to 700 mM NaCl. The injection volume was 3 ml and the diameter of the column was 12 mm and length 3 mm. The isolated fractions were monitored for protein content and PME activity. The fraction with the targeted enzyme, which showed NaCl independent activity, was further purified and concentrated by ultrafiltration and finally purified by a second semi-preparative cation-exchange chromatography step using a CIM carboxymethyl (CM) disk monolithic column consisting of two disks and applying a step gradient. From 1 kg of fresh tomato fruits, 7.5 mg of purified PME with molecular mass estimated to be 26 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was obtained. A fraction with mixed PME and polygalacturonase activity was also obtained. Compared to the published procedures for the isolation and purification of PME from plant materials, this new procedure is much faster and more efficient. The potential application of CIM disk short monolithic columns in the analysis and semi-preparative extraction and isolation of the PME isoenzyme is presented.