Purification of human placental prostaglandin 15-hydroxydehydrogenase

The NAD(+)-linked prostaglandin 15-hydroxydehydrogenase, which is responsible for the physiological inactivation of prostaglandins by catalysing the first step in the catabolism, was isolated and purified 995-fold from human placenta. The introduction of two new chromatographic steps in the purification procedure is responsible for an achieved specific activity of 1791 mU/mg. The molecular mass of the enzyme, as estimated by fast protein liquid chromatography, was 24,500 dalton. Sodium dodecyl sulphate discontinuous gel electrophoresis of the denatured enzyme revealed a molecular mass of 24,000 dalton. These data suggest that the enzyme consists of a single polypeptide chain.     

植物白头翁毒蛋白的分离、纯化及其组分测定

植物白头翁（ａｍｅｎｏｎｅ）茎的抽提液经ＣＭ－ＳＦＦ柱和ＳｅｐｈａｃｒｙｌＳ－２００柱分离纯化,得到一种毒蛋白,用高效凝胶蛋白柱和反相高效液相色谱法结合光电二极管阵列检测器确认分离峰的纯度,在高效凝胶蛋白柱上制备了少量毒蛋白纯样,测定了蛋白分子量和氨基酸组成。     

Use of chromatofocusing for separation of beta-lactamases. IX. Analytical chromatofocusing for the separation of a chromosomal cephalosporinase from Proteus vulgaris 1028

Simultaneous purification and isoelectric point (pI) determination was carried out at analytical scale of the chromosomal cephalosporinase from the Proteus vulgaris 1028 strain. Comparison of the enzyme to the purification results with m-aminophenylboronic acid-agarose affinity chromatography with sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that minute amounts of accompanying proteins having identical pI values but different molecular masses were found in the chromatofocused preparation. The molecular mass of the enzyme was 24,000 dalton. The pI was found to be 8.3.     

Rapid,High-Yield Purification of Rat Liver Glutathione Peroxidase by High Performance Liquid Chromatography

Abstract

Glutathione peroxidase (GSH:H₂O₂ oxidoreductase, EC 1.11.1.9) was purified 3500-fold from rat liver with a yield of 42% using high performance liquid chromatography. The crucial purification step was size-exclusion chromatography on a Spherogel TSK-3000SW column, and the purified enzyme eluted as a single peak. The enzyme stained as a single band following SDS-gel electrophoresis. The molecular weight of the enzyme was estimated to be 105,000, and the subunit molecular weight determined by SDS-gel electrophoresis was 25,000. Polyacrylamide gel electrophoresis indicated five bands of protein with a broad of enzymatic activity. Isoelectric focusing resulted in a peak of enzymatic activity at pH 6.9 with a shoulder at pH 7.3. The specific activity of the purified enzyme was 1,100 μmol of NADPH oxidized per minute per milligram of protein.     

Purification and Characterization of a Urethanase from Penicillium variabile

Urethanase produced by Penicillium variabile was purified through ultrasonication, concentration by polyethylene glycol 20,000, and Superdex G-200 gel filtration chromatography. The molecular weight of urethanase was determined to be around 96 kDa by gel filtration. The purified enzyme showed a single band in SDS-PAGE with the molecular weight of ~13.7 kDa, which suggests that the enzyme has a multimeric structure composed of the same subunits. Peptide map fingerprinting analysis was then carried out by MALDI/TOF-TOF MS. Within the known sequences in NCBI, glucosamine-6-phosphate deaminase and 6-phosphogluconate dehydrogenase get high score as compared with urethanase. Sequence analysis informs that N-terminal sequence of urethanase is GTNTADNDAA. The Minchaelis constant (K _m) and maximum reaction rate (V _m) of urethanase are 27.2 mmol/L and 156.25 μmol/L min, respectively.     

Purification and characterization of hamster hepatic microsomal N,O-acetyltransferase.

A microsomal N,O-acetyltransferase which activates carcinogenic arylacetohydroxamic acids was purified 75-fold from hamster liver sequentially by anion exchange column chromatography, chromatofocusing, gel filtration, and hydroxyapatite column chromatography. The purified enzyme, AT-2, was a glycoprotein with a molecular weight of 60000 and a pI value of 5.4. The N-terminal amino acid sequence of AT-2 was: 60000 and a pI value of 5.4. The N-terminal amino acid sequence of AT-2 was: Asp-Ser-Pro-Ser-Pro-Ile-Arg-Asn-Thr-His-Thr-Gly-Gln-Val-Arg-Gly-Leu-Val- His- Lys-. This sequence was highly homologous to that of the form 2 carboxylesterase of rabbit liver, but not to that of major hepatic microsomal carboxylesterases of hamster and other species. AT-2 catalyzed the hydrolysis of 4-nitrophenyl acetate and the N,O-acetyltransfer of N-hydroxy-2-acetylaminofluorene. Both enzyme activities were strongly inhibited by paraoxon, but not by iodoacetamide. These results demonstrate that this N,O-acetyltransferase is a member of carboxylesterase (EC 3.1.1.1).     

猪精浆中附睾特异性蛋白质的纯化及其一级结构的研究

哺乳动物体中附睾内精子的成熟过程要经过配子融合而形成合子的过程,其中,精子聚合到附睾分泌的特异蛋白质上的过程是合子形成的重要环节,目前已证明附睾中分泌出来的一些特异蛋白质和精子成熟之间的相互关系,但精子动能的获得或与合子的结合能力的研究尚不十分清楚。     

Presence of the basement membrane component--heparan sulfate proteoglycan--in bovine lens capsules

Heparan sulfate proteoglycan was extracted from bovine lens capsules by 0.45 M NaCl/2 M urea and purified using ion-exchange chromatography and gel filtration. The proteoglycan was found to consist of protein and carbohydrate in a ratio of 75 to 25. The estimated average molecular weight of the heparan sulfate proteoglycan eluted by 0.2 M NaCl on a diethylaminoethyl (DEAE)-cellulose column was 400 kilodaltons (kDa) and that of its glycosaminoglycan was 18.8 kDa. The amino acid composition of the proteoglycan was quite similar to that of the bovine glomelular basement membrane.     

Application of high-performance liquid chromatography to the purification, disintegration and molecular mass determination of pyruvate dehydrogenase multi-enzyme complexes from different sources

The pyruvate dehydrogenase complex is associated with the inner mitochondrial membrane. A gentle and rapid purification procedure, especially for the very unstable pyruvate dehydrogenase complex from the extremely thermophilic organism Thermus aquaticus, is described. This procedure is based essentially on a combination of hydrophobic interaction and of adsorption chromatography by the rapid fast protein liquid chromatographic technique. Applying the same method, a relative molecular mass of 9.1 . 10(6) daltons was obtained by gel filtration on Superose 6 HR 10/30 for the pyruvate dehydrogenase complex from T. aquaticus. The same column served to resolve the pyruvate dehydrogenase complex into its enzyme components.     

Resolution and quantitation of apolipoproteins A-I and A-II from human high-density lipoprotein by size exclusion high-performance liquid chromatography

Apolipoproteins A-I and A-II, extracted from human high-density lipoprotein (HDL), were resolved and quantified by size exclusion high-performance liquid chromatography on TSK 125 and TSK 250 analytical columns connected in series without the use of chemical denaturants or detergents in the eluent buffer. The columns were pre-equilibrated with a solution containing 0.1 M sodium phosphate, pH 7.2, 0.2 M sodium chloride at a flow-rate of 1 ml/min. Delipidated HDL (1 mg protein per ml) was resolved into two populations of apolipoprotein (apo) A-I: one representing the apo A-I monomer and the other, a self-associated form with a molecular weight of approximately 120,000 daltons. The column eluates were screened for immunoreactivity to apo A peptides, and the identity of each peak was confirmed by sodium dodecyl sulfate--polyacrylamide gel electrophoresis followed by immunoblot analysis. Apo A-I peptides isolated by high-performance liquid chromatography disrupted unilamellar phospholipid vesicles to form smaller phospholipid particles that eluted on gel filtration columns within the size range of HDL. Thus, a rapid method for the isolation and quantitation of non-denatured apolipoproteins from HDL has been developed using size exclusion high-performance liquid chromatography.     

High Performance Liquid Chromatography of Proteins on a Hydroxyapatite Column

Abstract

High performance liquid chromatography on a spherical ceramic type hydroxyapatite has been applied successfully for the separation of various kinds of proteins. Twenty-one proteins of various origin, having an isoelectric point of 3.3–11.0 and a molecular weight of 11,000–190,000 daltons, were loaded on the column and eluted by linear gradient of sodium phosphate at pH 6.8. The chromatography showed good resolution and high recovery for the proteins. The analysis of the retention behavior, relation between capacity ratio and physicochemical properties of proteins, showed a tendency that the capacity ratio of protein increased with the pI value of the protein.     

Chromatofocusing nonporous reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry of proteins from human breast cancer whole cell lysates: a novel two-dimensional liquid chromatography/mass spectrometry method

A novel two-dimensional two-column liquid chromatography/mass spectrometry (LC/MS) technique is described in this work, where chromatofocusing (CF) has been coupled to nonporous reversed-phase (NPS-RP) HPLC to separate proteins from human breast epithelial whole cell lysates. The liquid fractions from NPS-RP-HPLC are readily amenable to direct on-line analysis using electrospray ionization orthogonal acceleration time-of-flight mass spectrometry (ESI-TOFMS). A key advantage of this technique is that proteins can be 'peeled off' in the liquid phase from the CF column according to their isoelectric points (pI) in the first chromatographic separation dimension. The NPS-RP-HPLC column further separates these pI-focused fractions based upon protein hydrophobicity as the second chromatographic dimension. The third dimension involves on-line molecular weight determination using ESI-TOFMS. As a result, this method has the potential to be fully automated. In addition, a 2-D protein map of pI versus molecular weight is generated, which is analogous to a 2-D gel image. Thus, this technique may provide a means to study differential expression of proteins from whole cell lysates.     

来源于类芽孢杆菌属碱性甲壳素酶的分离纯化及其性质

甲壳素,又名几丁质(chitin),是自然界中含量仅次于纤维素的第二大天然多糖,有第六生命要素之美称.其主要存在于甲壳类动物的外壳、真菌细胞的细胞壁以及一些昆虫的外壳中,每年自然界中约有100多亿吨甲壳素生成.甲壳素是由2-乙酰氨基-2-脱氧-D-吡喃葡萄糖和2-氨基-2-脱氧-D-吡喃葡萄糖通过β-1,4糖苷键连接而成的二元线性聚合物,分子链中分布许多羟基、氨基及乙酰氨基,形成大量分子间及分子内氢键,致使其结晶度较高,化学性质十分稳定,直接利用较为困难.甲壳素不溶于稀酸、稀碱以及一般有机溶剂,工业上常用强酸强碱法处理甲壳素,以制备壳寡糖类产品,但该方法具有产品结构不单一,环境污染较为严重等缺点.甲壳素酶可特异性水解甲壳素链中β-1,4糖苷键,得到甲壳寡糖和N-乙酰氨基葡萄糖.酶解法降解甲壳素工艺简单、反应条件温和、环境友好,有很好的应用前景.我们以Paenibacillus pasadenensis CS0611为出发菌株,以蟹壳粉末为培养基唯一碳源及氮源,在适宜条件下培养48 h.发酵液经离心、硫酸铵(80％饱和度)盐析、透析除盐后得到粗酶液.再利用HiTrap DEAE FF离子交换层析和HiLoad 26/600Superdex 200 pg凝胶过滤层析对该粗酶液进行分离纯化,以得到电泳纯甲壳素酶.所制备甲壳素酶比活力为10.28 U/mg,最终纯化倍数为5.3,酶活得率为15.7％.SDS-PAGE结果表明,该甲壳素酶相对分子质量约为69 kDa.后经MALDI-TOF-MS鉴定,该酶部分肽段和来源于另一株Paenibacillus pasadenenss的甲壳素酶(accession No:gi655151624)具有较高的同源性,进一步证实所纯化蛋白为甲壳素酶.对上述纯化的甲壳素酶的酶学性质进行研究,结果发现:其最适反应温度为50℃,在20-35℃内有较好的稳定性,50℃及以上热稳定性较差;最适pH为5.0,在pH4.0-11.0间具有较高稳定性,表明该酶具有很好的耐碱性;金属离子对该酶催化活性没有明显的激活作用,表明该甲壳素酶是非金属酶.同时,对该酶的底物特异性进行研究,发现该酶对胶体甲壳素和甲壳素水解能力较强,对淀粉和纤维素无水解能力,对不同脱乙酰度的壳聚糖的水解程度随脱乙酰度不同而变化,表明该酶只能特异性识别并降解GlcNAc-GlcNAc之间的糖苷键;以胶体甲壳素为底物时,米氏常数Km为4.41 mg/mL,最大反应初速度为1.08 mg/min.利用薄板层析和高效液相色谱对酶解产物进行分析,结果表明该甲壳素酶对胶体甲壳素的降解产物主要是(GlcNAc)2.综上所述,本研究所涉甲壳素酶在甲壳二糖的酶法制备方面具有较好的应用前景.     

Purification of glutathione reductase from chicken liver and investigation of kinetic properties

Glutathione reductase was purified from chicken liver and some characteristics of the enzyme were investigated. The purification procedure was composed of four steps: preparation of homogenate, ammonium sulfate precipitation, 2′,5′-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. Owing to the four consecutive procedures, the enzyme was purified 1714-fold, with a yield of 38%. Specific activity at the final step was 120 enzyme unit (EU)/mg of protein. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the enzyme was found to be 100 kDa by Sephadex G-200 gel filtration chromatography, and the subunit molecular weight was found to be 43 kDa by SDS-PAGE. Optimum pH, stable pH, optimum ionic strength, and optimum temperature were 7.0, 7.4, 0.75 M Tris-HCl buffer including 1 mM EDTA, and 50°C, respectively. K _M and V _max values for NADPH and glutathione disulfide (GSSG) substrates were also determined for the enzyme.     

Biochemical Characterization of Novel Bioactive Protein from Silkworm (Bombyx mori L) Fecal Matter

In this study, complete purification and biochemical characterization of protein is presented. The protein was purified by using Sephadex G-75 gel filtration column followed by reverse-phase high-performance liquid chromatography in a C18 column. The molecular weight of the protein was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, mass spectrum matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) and liquid chromatography-electrospray ionization tandem mass spectrometry. Protein was fragmented by trypsin based on the m/z values obtained by MALDI-TOF-MS analysis. The peptide fragments sequence showed homology with DEAD-box-ATP-dependent RNA helicase 45, present in a public domain, National Centre for Biotechnology Information. The protein exhibited antibacterial activity against selected Gram +/? bacteria. The analgesic activity was determined by conducting acetic-acid-induced writhing test in mice.     

Separation of proteins by consecutive sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and reversed phase high performance liquid chromatography

A procedure for the preparative separation of proteins was developed by using consecutively sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) and reversed phase high performance liquid chromatography (HPLC). The proteins were separated by SDS-PAGE and afterwards extracted from the gel. The extracted proteins were separated from SDS and other small molecular weight contaminants on a Fractogel TSK HW-40 (F) column in acidic aqueous acetonitrile. The proteins eluted from the Fractogel column were fractionated by HPLC. The identity and purity of the recovered proteins was confirmed by SDS-PAGE analysis.     

Purification and Characterization of a Cu,Zn-SOD from Garlic (Allium sativum L.). Antioxidant Effect on Tumoral Cell Lines

Crude garlic extract contains one Mn-superoxide dismutase designated as SOD1 and two Cu,Zn superoxide dismutases as SOD2 and SOD3. The major isoform SOD2 was purified to homogeneity by Sephacryl S200-HR gel filtration, DEAE Sepharose ion exchange chromatography, and chromatofocusing using PBE 94. SOD2 was purified 82-fold with a specific activity of 4,960 U/mg protein. This enzyme was stable in a broad pH range from 5.0 to 10.0 and at various temperatures from 25 to 60°C. The native molecular mass of SOD2 estimated by high performance liquid chromatography on TSK gel G2000SW column was 39 kDa. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis showed a single band near 18 kDa, suggesting that native enzyme was homodimeric. The isoelectric point as determined by chromatofocusing was 5. Analysis of its N terminal amino acid sequence revealed high sequence homology with several other cytosolic Cu,Zn-SODs from plants. Exposure of cancer cell lines to garlic Cu,Zn-SOD2 led to a significant decrease in superoxide content with a concomitant rise in intracellular peroxides, indicating that the enzyme is active in mammalian cells and could, therefore, be used in pharmacological applications.     

凝胶层析和离子交换层析结合法纯化高盐发酵液中谷氨酰胺转胺酶

采用凝胶层析和离子交换层析相结合的方法分离纯化高盐培养基中的谷氨酰胺转胺酶,优化的凝胶层析的条件,上样量6mL,流速为0.25 mL/min;离子交换层析的上样量50 mL,流速为3 mL/min.酶被纯化了4.22倍,比活力达17.33 U/mg蛋白,回收率为77.5％.液相色谱-串联质谱鉴定、蛋白质数据库比对结果表明,纯酶与AAN01353是同种蛋白质.     

The complete amino acid sequence of an abortifacient protein, karasurin

The complete amino acid sequence of a new abortifacient protein, karasurin, was determined. Karasurin, which was isolated from fresh root tubers of Trichosanthes kirilowii Maximowicz var, japonicum Kitamura (Cucurbitaceae), was a highly basic protein with pI 10.1 and molecular weight of 28,000. Intact karasurin was cleaved with cyanogen bromide, lysyl endopeptidase, formic acid and 2-(2'-nitrophenyl-sulfenyl)-3-methyl-3-bromoindolenine (BNPS-skatole), respectively. Cleavages with N-bromosuccinimide (NBS), trypsin and pepsin were performed for the fragments. The resultant peptide fragments were separated by gel filtration chromatography, reversed-phase high performance liquid chromatography (HPLC) or gel filtration HPLC following sequence analyses by automated Edman methods. Karasurin consists of 246 or 247 amino acid residues with a calculated molecular weight of 27,144 or 27,215 differing only at the C-terminus with the addition of alanyl residue. Two C-terminal sequences were identified as Asn-Asn-Met-OH and Asn-Asn-Met-Ala-OH by sequence analyses and hydrazinolysis, but there was no micro-heterogeneity in other peptides analysed. The sequence of karasurin revealed a considerable similarity to that of trichosanthin and alpha-trichosanthin, which are known as abortifacient, ribosome-inactivating and anti human immunodeficiency virus (HIV) (the virus causing acquired immunodeficiency syndrome (AIDS) proteins, with 93% and 98% identity, respectively.     

Novel method of purification of human leukocytic elastase using adsorption on a high-performance liquid chromatography gel permeation column

Neutrophil elastases are serine proteinases released during acute and chronic inflammatory states. We have developed a novel isolation method for neutrophil elastase, involving conventional gel chromatography followed by adsorption of protein at low ionic strength on a high-performance liquid chromatography gel permeation column. The bound elastase is then eluted by application of higher ionic strength. This adsorption step at low ionic strength, a step to be avoided in most purification methods, was used to advantage here to allow isolation of homogeneous material. This purification procedure should be useful for quick, simple bulk preparation of the enzyme.