一种核糖核酸酶构象稳定性的研究

Aspergillus niger SA-13-20核糖核酸酶是从突变株A.niger SA-13-20分泌的胞外酶中分离出的一种新的核糖核酸酶。采用紫外光谱、荧光光谱和红外光谱研究了A.niger SA-13-20核糖核酸酶在不同pH值条件下的构象稳定性。紫外光谱和荧光光谱结果均表明该酶蛋白在酸性和弱碱性pH值下构象较稳定,当pH值高于9.6时构象不稳定;红外光谱结合去卷积和曲线拟合技术对蛋白质酰胺Ⅰ带的测定和处理结果表明,在室温下,pH 5.0时该酶蛋白二级结构中α-螺旋、β-折叠、转角和无规结构所占的成分分别为13.28%、42.30%、26.48%和17.95%。测得该酶的热解链温度Tm、解链熵变ΔSm及解链焓变ΔHm分别为70.1℃、644 J.mol-1.K-1及22.1 kJ.mol-1,表明该酶属于耐热能力较强的核糖核酸酶。研究结果有助于揭示该酶结构与功能的关系,推动其在科研和生产等方面的应用。     

Purification and properties of bilirubin oxidase fromMyrothecium verrucaria

Bilirubin oxidase was purified from a culture filtrate of Myrothecium verrucaria Mv 2, 1089 by DEAE-cellulose and Sephadex G-100 column chromatographies. The purified enzyme had a specific activity of 30 U/mg protein and showed a single band on polyacrylamide gel electrophoresis. Some of the general properties of this bilirubin oxidase were as follows: the optimum pH for the enzyme reaction was 7.5 and the optimum temperature was 50 degrees C. The enzyme was stable at pH ranging from 9.0 to 9.5. The mol wt was calculated to be 61,900-62,700 by SDS-PAGE and gel-filtration technique. The apparent Km value of the bilirubin oxidase was calculated to be 9.4 x 10(-5) mol/L. The enzyme activity was greatly reduced by incubation of bilirubin oxidase with Fe2+, Hg+, NaN3, NH+4, and Zn2+. The enzyme reaction was inhibited in the presence of Ca2+, Hg+, Zn2+, Fe2+, and BSA.     

铊(I)离子选择电极动力学分析法测定超痕量铁

Fe(II) induces the reaction between Tl3+ and H2O2. The rate of reaction is linearly proportional to the concentration of Fe2+ in the range 2.5 ?10-9-2.5 ?10-8 mol dm-3 (20? and 5 ?10-9-5 ?10-8 mol dm-3 (15?. The standard deviation is less than 0.071 ?10-8. A 1000-fold excess of Zn2+, Cd2+, Mg2+, Ni2+, Pb2+, Ba2+, Ca2+, Li+, Na+, Ag+, NO3-, SO42-, AcO-, HPO42-, 500-fold excess of Al3+, Fe3+, Co2+, Hg2+ and 100-fold excess of Ti4+, Cr3+, Cu2+, Br-, Cl- can be tolerated, but reducing agents such as (NH2)2SO4, NH2OH.HCl interfered. This kinetic method was applied to determine Fe(II) in standard zinc sample and fountain water, with satisfactory results.     

Purification and Characterization of DNA Methylase From HL-60 Cells

A solid leukemia sarcoma has been successfully developed after subcutaneous inoculation of the cultured human promyelocytic leukemia cells (HL-60 cells) into unde mice. The solid leukemia sarcoma is a more plantiful source than the cultured cells for enzymatic study and its growing environment is closer to that of the human body than the cultured cells.We establish an efficient procedure of purifying HL-60 cells DNA methylase which includes: disruption of HL-60 cells by homogenization and sonication, removing the cell fragments and cellular particles by centrifuge and ultracentrifuge (105.000 g); removing endogenous DNA by streptomycin sulfate, salting out by (NH4)2SO4, ion exchange chromatography on DEAE-cellulose (DE-52), gel filtration over Sephadex G-100 column.The DNA methylase from HL-60 cells has been purified 204 fold by this procedure. The purified enzyme shows a single-band on PG-PAGE. A 479-kD molecular weight of this enzyme is measured by PG-PAGE. The enzyme properties of HL-60 DNA methylase     

双水相萃取结合液相色谱法分离蛋白质

建立了PEG/( NH4)2SO4双水相体系萃取富集,结合液相色谱分离分析多种蛋白质的方法.考察了无机盐种类和浓度、PEG分子量、pH值和温度等因素对双水相形成以及对细胞色素C、肌红蛋白、牛血清白蛋白、溶菌酶、胰蛋白酶分配行为的影响.结果表明,上述5种蛋白在室温、pH 3.5～9.0范围内,可在15％ PEG-4000/10％ (NH4)2SO4双水相体系中得到富集,且主要集中在下相.同样条件下,血清中的高丰度蛋白在上下相均有分配,下相分配量较大.通过双水相萃取分离蛋白质及对液相色谱一定时间段的色谱峰收集,可初步实现血清中高丰度蛋白质的分离去除.     

离子色谱-伏安极谱法联用同时测量空气中阴阳离子及重金属含量

通过空气样品液化器中真空泵的作用,空气转化为液体,从而能持续提供样品流,以供离子色谱仪和伏安极谱仪检测。阐述了将空气样品液化器(PILS)与伏安极谱仪(VA)、离子色谱仪(IC)联机分析空气中气溶胶的方法,其中伏安极谱仪可以分析空气中的Zn、Cd、Pb、Cu等重金属含量,离子色谱仪可以分析空气中的Cl-、NO2-、NO3-、SO42-、Li+、Na+、NH4+、K+、Ca2+、Mg2+等阴、阳离子的含量。方法取得了很好的精密度与准确度。     

Partial purification and characterization of epidermal plasminogen activator and their inhibitor

Plasminogen activator (PA) and PA inhibitor were partially purified from 2-d-old rat epidermis and characterized in order to elucidate the enzyme-inhibitor interaction in epidermis. PA extracted with buffer containing KSCN was first purified by Blue-Sepharose affinity chromatography. Separation of two PAs, with relative molecular mass (Mr) of 66000 and 44000, was accomplished by Con A-Sepharose column chromatography. The Mr 66000 enzyme had the properties of tissue-type PA (t-PA), while the Mr 44000 enzyme showed those of urokinase-type PA (u-PA) as determined by immunological and fibrin-binding studies. PA inhibitor was extracted in 1,4-piperazinediethanesulphonic acid buffer and purified by (NH4)2SO4 precipitation, gel filtration followed by a Mono Q column in an FPLC system. This inhibitor showed Mr 60000 and inhibited human u-PA activity in a dose- and time-dependent manner, but did not inhibit the activity of t-PA from human and murine melanoma cells or plasmin. It inhibited epidermal PA, Mr 44000, more effectively than it did the Mr 66000 epidermal PA. It was stable at 60 degrees C for 60 min or between pH 5 and 11. This study indicates that both u-PA and t-PA function in normal rat epidermis. On the other hand, an inhibitor which preferentially acts against u-PA exists, but inhibitor to t-PA does not appear to operate under normal epidermal functions.     

High-resolution determination of H+ by ion chromatography. Application to the simultaneous determination of H+, Na+, NH4+ and K+ in acid rain.

An ion chromatographic (IC) method was developed for the high-resolution determination of a sample's free hydrogen ion concentration (H+). Highly purified lithium dodecyl sulfate was used as the stationary phase, a slightly acidified aqueous LiCl solution was used as the mobile phase and conductivity was used for analyte detection. An electrical double layer (EDL) containing H+ was established on the stationary phase by using a slightly acidified electrolyte solution as the eluent. H+ in the EDL protonated any weak acid groups (i.e., silanols) on the stationary phase so that H+ from the sample could be retained/separated purely by dodecyl sulfate. The optimum molar ratio of H+:Li+ in the EDL for this IC system was obtained by using an aqueous solution containing 40.0 mM LiCl and 0.07 mM H2SO4 as the eluent. After separation, H+ was detected by direct conductimetric measurement. An H+ detection limit of better than 8.2 x 10(-6) M was obtained from the analysis of standard aqueous H2SO4 solutions. Other monovalent cations could also be separated with this method, giving detection limits of 7.4 x 10(-5), 4.3 x 10(-5) and 4.2 x 10(-5) M for Na+, NH4+ and K+, respectively. The method was applied to the simultaneous determination of H+, Na+, NH4+ and K+ in acid rain. The results obtained showed a significant improvement in reproducibility when compared with those from a conventional pH-meter. Acid rain samples with a pH < 5 could be analyzed with this IC system.     

Partial purification, and some properties and reactivities of cetraxate benzyl ester hydrochloride-hydrolyzing enzyme

Debenzylating enzyme from Aspergillus niger enzyme (commercial crude cellulase) catalyzes the hydrolysis of cetraxate benzyl ester hydrochloride (2), a precursor of the antiulcer agent (1). The enzyme was highly purified by three kinds of chromatographies (hydrophobic, ion exchange, gel filtration) with a recovery of 36%. The content of the debenzylating enzyme was about 0.1% in the crude cellulase, but the enzyme showed no cellulase activity. The purified enzyme was inactivated by Hg2+, and diisopropyl phosphorofluoridate (DFP). It was a monomer with a molecular weight of about 35,000, and its isoelectric point was estimated to be 5.3. It showed a debenzylating activity for the phenylpropionic acid benzyl ester moiety of various benzyl ester derivatives, and the benzyl ester of phenylalanine or that of tyrosine was also well hydrolyzed.     

芳香酸淋洗液的单柱阳离子色谱法测定碱金属,铵离子和烷基胺

研究了邻苯二甲酸、苯甲酸和对羟基苯甲酸３种芳香酸分别做淋洗液的单柱阳离子色谱法分离测定Ｌｉ＋、Ｎａ＋、ＮＨ４＋、Ｋ＋、甲胺、乙胺和正丙胺７种物质。３种芳香酸做淋洗液均可将７种物质分离开，且分离结果差异不大。在淋洗液浓度相同的条件下，邻苯二甲酸做淋洗液测定的检出限较低。选择３．０ ｍｍｏｌ／Ｌ邻苯二甲酸做淋洗液测得了上述７种物质的检出限和线性范围，并进行了叶面肥试样分析，７种物质的加标回收率在９６．４％～９８．６％之间。     

Alkaline ribonuclease associated with polyribosomes in fibroblasts of experimental granulation tissue.

Alkaline ribonuclease (RNase) from polyribosomes derived from experimental granulation tissue has been purified 1900-fold through affinity chromatography. The preparation was homogeneous in sodium dodecyl sulfate (SDS) polyacrylamide-gel electrophoresis with an estimated molecular weight of 15 000. Purified RNase was completely inhibited in the presence of divalent ions Mg2+(100 mM) and Ca2+(100 mM) but activated slightly with Na+(50 mM). The enzyme is an endonuclease and the best substrates were poly(U), mixed RNA from yeast, rRNA from granulation tissue and poly(C). The estimated apparent Km-values were 0.037, 0.064, 0.13 and 0.27 g1-1, respectively. In polyribosomes RNase occurred in both free and p-chloromercuribenzoate (pCMB)-liberated forms. The total activity was at the highest but the proportion of the free activity minimal in the granulation tissue during the maximal synthesis of collagen.     

醇与离子液体二元双水相体系萃取盐酸多西环素

基于小分子醇双水相体系和离子液体双水相体系,建立了正丙醇与亲水性离子液体1-丁基-3-甲基咪唑四氟硼酸[Bmim]BF4和(NH4)2SO4形成的二元双水相体系萃取盐酸多西环素的新方法。考察了(NH4)2SO4含量、正丙醇用量、pH值、离子液体含量以及盐酸多西环素含量对盐酸多西环素分配行为的影响。结果表明:当醇和离子液体二元双水相体系的pH值在4.0～5.0范围内,(NH4)2SO4含量为34%,且盐酸多西环素的质量浓度在25～95 mg/L之间时,该体系对盐酸多西环素的萃取率可达90.26%～95.71%,分配系数可达62.452～149.401。     

Simultaneous ion chromatographic separation of anions and cations on poly(aspartic acid) functionalized silica

Poly(aspartic acid)-silica (PolyCAT A), originally designed for the cation-exchange chromatography of proteins, is proposed for the simultaneous ion chromatographic separation of inorganic anions and cations. This is possible owing to the zwitterion-exchange properties of this stationary phase, which are attributed to the presence of both protonated aminopropyl and dissociated carboxylic groups in poly(aspartic acid) attached to the silica. The retention of alkali metal (Li+, Na+, K+), alkaline earth metal (Mg2+, Ca2+), ammonium and inorganic anions (Cl-, H2PO4-, Br-, NO2-, I-, IO3-, NO3-, ClO4-, SCN-) was tested in aqueous solutions of sulfuric, perchloric, sulfosalicylic, citric, oxalic, maleic and aspartic acids with conductimetric detection. The effect of eluent pH, together with the concentration and characteristics of the eluting ions, were studied. Under optimum conditions (0.3 mmol dm(-3) H2SO4-0.2 mmol dm(-3) Li2SO4 eluent), the simultaneous separation of three anions (Cl-, H2PO4-, NO3-) and four cations (Na+, K+, Mg2+, Ca2+), on a PolyCAT A column (200 x 4.6 mm id, 5 microm film thickness) was achieved in 9 min.     

Purification and characterization of an extracellular β-glucosidase with high transglucosylation activity and stability from Aspergillus niger No. 5.1

An extracellular beta-glucosidase was extracted from the culture filtrate of Aspergillus niger No. 5.1 and purified to homogeneity by using ammonium sulfate precipitation, Chitopearl-DEAE chromatography, and Sephadex G-100 chromatography. The specific activity of the enzyme was enriched 6.33-fold, with a recovery of 11.67%. The enzyme was a monomer and the molecular mass was 67.5 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 66.5 kDa by gel-filtration chromatography. The enzyme had optimum activity at pH 6.0 and 60 degrees C and was stable over the pH range of 3.0-9.0. It showed specificity of hydrolysis for p-nitrophenyl-beta-D-glucoside and cellobiose. The Km and Vmax values of the enzyme for cellobiose and salicin were 5.34 mM, 2.57 micromol/(mL.s), and 3.09 mM, 1.34 micromol/(mL.s), respectively. Both amino acid composition and N-terminal amino acid sequence of the enzyme were determined, which provides useful information for cloning of this enzyme.     

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 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.     

Purification and properties of cyclodextrinase fromBacillus stearothermophilus HY-1

A thermostable cyclodextrinase (EC 3.2.1.54) fromBacillus stearothermophilus HY-1 was purified to homogeneity by disc-electrophoresis after sonication disruption, ammonium sulfate fractionation, DEAE-cellulose(DE32) column chromatography, hydroxyapatite chromatography, Sephadex G150 gel-filtration, and α-cyclodextrin-AH-Sepharose 4B affinity chromatography. The enzyme was purified 230-fold with 21.2% of activity recovery. The optimal substrates of the enzyme were α-, Β-, and γ-cyclodextrins and linear maltooligosaccharides, and the final product was mainly maltose. The enzyme could hydrolyze pullulan to produce panose. It could also hydrolyze soluble starch, amylose, and amylopectin, but not glycogen. The Km and Vmax for α-, Β-, and γ-cyclodextrins were 1.79, 1.67, and 2.50 mg/mL, and 336, 185, and 208 Μmol/mg/min, respectively. The molecular weight of the enzyme was 61,000 by SDS-gel-electrophoresis. The isoelectric point was pH 5.0. The enzyme was most active at pH 6.2 and 55‡C, and it was strongly inhibited by Cu²⁺, Hg²⁺, Zn²⁺, Pb²⁺, and slightly by Fe²⁺. The effect of some protein modification reagents on the activity of the enzyme suggested that tryptophan and histidine residue(s) may be located at the active site. The amino acid composition of the enzyme was also determined.     

离子色谱法测定饲料中氯化胆碱和三甲胺的含量

建立了离子色谱法测定饲料中氯化胆碱含量及鉴别饲料中氯化胆碱及掺假物三甲胺的方法。选用IonPac CS12阳离子交换色谱柱(250 mm×4 mm i.d.)和8.5 mmol/L H2SO4淋洗液,抑制型电导检测,在16 min内分离测定了包括胆碱和三甲胺在内的8种阳离子。胆碱和三甲胺的最小检出限分别为0.1 mg/L和0.05 mg/L。方法回收率为99.25%~102.5%。该方法具有灵敏度高、选择性强、操作简单等优点。     

Synthesis and characterization of octa- and hexanuclear polyoxomolybdate wheels: role of the inorganic template and of the counterion

Eight new compounds based on [O3PCH2PO3]4- ligands and {MoV2O4} dimeric units have been synthesized and structurally characterized. Octanuclear wheels encapsulating various guests have been isolated with different counterions. With NH4+, a single wheel was obtained, as expected, with the planar CO32- guest, (NH4)12[(MoV2O4)4(O3PCH2PO3)4(CO3)2].24H2O (1a), while with the pyramidal SO32- guest, only the syn isomer (NH4)12[(MoV2O4)4(O3PCH2PO3)4(SO3)2].26H2O (2a) was characterized. The corresponding anti isomer was obtained with Na+ as counterions, Na12[(MoV2O4)4(O3PCH2PO3)4(SO3)2]39H2O (2b), and with mixed Na+ and NH4(+) counterions, Na+(NH4)11[(MoV2O4)4(O3PCH2PO3)4(SO3)2].13H2O (2d). With [O3PCH2PO3]4- extra ligands, the octanuclear wheel Li12(NH4)2[(MoV2O4)4(O3PCH2PO3)4(HO3PCH2PO3)2].31H2O (4a) was isolated with Li+ and NH4+ counterions and Li14[(MoV2O4)4(O3PCH2PO3)4(HO3PCH2PO3)2].34H2O (4c) as a pure Li+ salt. A new rectangular anion, formed by connecting two MoV dimers and two MoVI octahedra via methylenediphosphonato ligands with NH4+ as counterions, (NH4)10[(MoV2O4)2(MoVIO3)2(O3PCH2PO3)2(HO3PCH2PO3)2].15H2)O (3a), and Li9(NH4)2Cl[(MoV2O4)2(MoVIO3)2(O3PCH2PO3)2]. 22H2O (3d) as a mixed NH4+ and Li+ salt have also been synthesized. The structural characterization of the compounds, combined with a study of their behavior in solution, investigated by 31P NMR, has allowed a discussion on the influence of the counterions on the structure of the anions and their stability. Density functional theory calculations carried out on both isomers of the [(MoV2O4)4(O3PCH2PO3)4(SO3)2]12- anion (2), either assumed isolated or embedded in a continuum solvent model, suggest that the anti form is favored by approximately 2 kcal mol(-1). Explicit insertion of two solvated counterions in the molecular cavity reverses this energy difference and reduces it to less than 1 kcal mol(-1), therefore accounting for the observed structural versatility.     

Determination of saccharides in biological materials by high-performance anion-exchange chromatography with pulsed amperometric detection

High-performance anion-exchange chromatography (HPAEC) coupled with pulsed amperometric detection (PAD) under alkaline conditions (pH 9-13) separates aminosaccharides, neutral saccharides and glycuronic acids based upon their molecular size, saccharide composition and glycosidic linkages. Carbohydrates were extracted by utilizing 0.5 M H2SO4 (neutral monosaccharides), 0.25 M H2SO4 coupled with enzyme catalysis (glycuronic acids) and 3 M H2SO4 (aminosaccharides). Solid-phase extraction with strong cation and strong anion resins was used to partition the cationic aminosaccharides and anionic glycuronic acids and to deionize acid extracts for neutral saccharides. Separation was conducted on a medium-capacity anion-exchange column (36 mequiv.) utilizing sodium hydroxide (5-200 mM and sodium acetate (0-250 mM) as the mobile phase. The saccharides were detected by oxidation at a gold working electrode with triple-pulsed amperometry. HPAEC-PAD was found superior to high-performance liquid chromatography with refractive index (RI) detection for neutral monosaccharides and aminosaccharides and to low-wavelength UV detection for glycuronic acids in terms of resolution and sensitivity. HPAEC-PAD was not subject to interferences as was the case for low UV detection (210 nm) or RI analyses and was highly selective for mono- and aminosaccharides and glycuronic acids. The use of HPAEC-PAD was applied for the determination of the saccharide composition of organic materials (plant residues, animal wastes and sewage sludge), microbial polymers and soil.