低分子量紫色酸性磷酸酶多克隆抗体的制备及应用

将最近从地瓜中提出来的低分子量紫色酸性磷酸酶（smPAP)基因克隆到GST融合蛋白表达载体pGEX－2T中，在大肠杆菌BL21codon plus中进行表达，用表达的融合蛋白免疫兔子产生多克隆抗体，用抗血清在昆虫细胞中表达的smPAP和地瓜提取液中分离纯化的PAP进行检测，产生了良好的交叉反应。     

紫色酸性磷酸酯酶模型化合物水解ATP的研究

紫色酸性磷酸酯酶（PAPS）是含铁的糖蛋白，能够从植物和动物体内分离出来，在微酸性条件下，能催化水解磷酸酯阻］，该类酶具双核铁中心活性部位，每个蛋白有两个铁原子，两个铁原子以ellelll形式存在时，具有催化活性·近年来，人们对研究磷酸酯水解酶感兴趣，是因为这个课题     

Inhibition of Model Compound of Purple Acid Phosphatases on Growth of Aerobacter aerogenes Investigated by Microcalorimetry

Ironisoneofthefourteentypesofessentiallybiologi caltraceelements .Ironplaysanimportantroleinlivingbodyanditwasregardedastherequiredtraceelementforlife .Manystudiesindicatethatdeficiencyorexcessofironmaybethecauseofmanydiseases .Furthermore ,ironisactivecenterofpigmentoxidase ,andsuccinicdehydroge nase ,etc .Consequently ,thestudyoftheinfluenceofironanditscompoundsonmicroorganismissignificantforun derstandinglifephenomenon .1Inhibitoryorantibioticactionofpurpleacidphos phatases (PAPs)hasnotbe…     

Rosmarinic acid determination using biomimetic sensor based on purple acid phosphatase mimetic

A new heterodinuclear Fe(III)Zn(II) complex which mimics the active site of the hydrolytic enzyme red kidney bean purple acid phosphatase was synthesized and characterized by IR, CHN and X-ray crystallographic analyses. This complex, [Fe^IIIZn^II(μ-OH)bpbpmp-CH₃](ClO₄)₂, containing the ligand (H₂bpbpmp-CH₃ = {2-[bis(2-pyridylmethyl)aminomethyl]-6-[(2-hydroxy-5-methylbenzyl) (2-pyridyl-methyl) aminomethyl]-4-methyl-phenol}) was employed in the construction of a biomimetic sensor and used in the determination of rosmarinic acid in plant extract samples. The response parameters and optimization of the biomimetic sensor design were evaluated. The best performance of this sensor was obtained for 75:15:10% (w/w/w) of the graphite powder:nujol:Fe(III)Zn(II) complex, 0.1 mol L⁻¹ phosphate buffer solution (pH 7.5), 1.19 × 10⁻⁴ mol L⁻¹ hydrogen peroxide with frequency, pulse amplitude, and scan increment at 30 Hz, 100 mV, and 0.6 mV, respectively. The rosmarinic acid concentration was linear in the range of 2.98 × 10⁻⁵ to 3.83 × 10⁻⁴ mol L⁻¹ (r = 0.9991) with a detection limit of 2.30 × 10⁻⁶ mol L⁻¹. This biomimetic sensor demonstrated long-term stability (300 days; 900 determinations) and reproducibility, with a relative standard deviation of 12.0%. The recovery study of rosmarinic acid in plant extract samples gave values from 90.3 to 98.3% and the concentrations determined showed agreement when compared with those obtained using capillary electrophoresis at the 95% confidence level.     

Biosynthesis of the Purple Membrane of Halobacteria

Halobacteria are extremely specialized organisms. They live exclusively in saturated solutions of common salt. The cell membrane of these bacteria exhibit insular regions which can be isolated by membrane fractionation. These regions consist of a lipid matrix containing bateriorhodopsin molecules in a hexagonal crystalline arrangement. Bacteriorhodopsin is a deep purple retinal-protein complex (“purple membrane”). The purple membrane functions as a light energy converter.—How can such a differentiated membrane region arise? In vivo studies on the biosynthesis of the purple membrane showed another cell membrane fraction, the so-called brown membrane, to be a biosynthetic precursor. Bacterioopsin (the retinal-free protein) is initially incorporated into the brown membrane and can only form the purple membrane by crystallization in an energy-dependent reaction after prior reaction with retinal. This reaction is reversible. Removal of the retinal by formation of retinal oxime causes the purple membrane regions to disappear. Reconstitution of the bacteriorhodopsin by addition of retinal regenerates the purple membrane.     

Immobilization of Bromocresol Purple in Inorganic-Organic Sol-Gel Thin Film with Presence of Anionic and Non-ionic Surfactants

Sol-gel materials known as organically modified silicates (ORMOSILs) offer interesting features such as chemical and mechanical stability. In this paper VTES (vinyltriethoxysilane) and TEOS (tetraalkoxysilane) are mixed in 3:1 ratio. Sol-gel solution was prepared by hydrolysis process of precursors by using ethanol as solvent. After a while a pH-sensitive indicator bromocresol purple (BCP) and surfactant were incorporate into the sol-gel mixture. The percentage of sodium dodecyl sulfate (SDS) and polyethylene glycol (PEG) which act as surfactant were varied to observe the effect of improving host material's nanostructure as well as the interaction between BCP and sol-gel matrices. The absorption peak of the BCP dye changed significantly in the presence of surfactant compared to pure VTES: TEOS mixture (control) in the range of 400 to 450 nm. The presence of BCP dye in the sol-gel mixture can be determined via FTIR spectrum with a =C–H stretch in aromatics observed at 3100-3000 cm^-1 which represented the aromatic of the BCP structure. The addition of BCP and surfactant had influenced the FTIR spectra of VTES: TEOS sol-gel materials. Addition of an anionic surfactant to the sol-gel mixtures was found to increase the BCP dye and the sol-gel matrices interaction, thus reducing the dye's tendency to leach. This work shows that sol-gel derived matrices yield dyes with sufficient rigid environment and addition of the surfactant helps to improve the interaction of filler matrices. The anionic SDS shows better leaching resistant compared to non-ionic PEG surfactant. Results of this study offer an attractive possibility to optimize the doped sol gel matrices to be used as sensing material in aqueous condition.     

Eutectic Media Open a Synthetic Route to Oligocitrazinic Acid Fluorophores of Purple Hue

Under isochoric and solvent-free conditions, the reaction between ammonium formate and citric acid results in a deeply purple reaction product with fluorescent properties. This brings this reaction in the realm of bio-based fluorophores and bottom-up carbon nanodots from citric acid. The reaction conditions are optimized in terms of UV–vis spectroscopic properties and, subsequently, the main reaction product is separated. While the structural analysis does not give any indication for carbon nanodots in a general sense, it points towards the formation of molecular fluorophores that consist of oligomerized citrazinic acid derivatives. Furthermore, EPR spectroscopy reveals the presence of stable free radicals in the product. We hypothesize that such open-shell structures may play a general role in molecular fluorophores from citric acid and are not yet sufficiently explored. Therefore, we believe that analysis of these newly discovered fluorophores may contribute to a better understanding of the properties of fluorophores and CND from citric acid in general.     

Indole- and Pyrazole-Glycyrrhetinic Acid Derivatives as PTP1B Inhibitors: Synthesis,In Vitro and In Silico Studies

Regulating insulin and leptin levels using a protein tyrosine phosphatase 1B (PTP1B) inhibitor is an attractive strategy to treat diabetes and obesity. Glycyrrhetinic acid (GA), a triterpenoid, may weakly inhibit this enzyme. Nonetheless, semisynthetic derivatives of GA have not been developed as PTP1B inhibitors to date. Herein we describe the synthesis and evaluation of two series of indole- and N-phenylpyrazole-GA derivatives (4a–f and 5a–f). We measured their inhibitory activity and enzyme kinetics against PTP1B using p-nitrophenylphosphate (pNPP) assay. GA derivatives bearing substituted indoles or N-phenylpyrazoles fused to their A-ring showed a 50% inhibitory concentration for PTP1B in a range from 2.5 to 10.1 µM. The trifluoromethyl derivative of indole-GA (4f) exhibited non-competitive inhibition of PTP1B as well as higher potency (IC₅₀ = 2.5 µM) than that of positive controls ursolic acid (IC₅₀ = 5.6 µM), claramine (IC₅₀ = 13.7 µM) and suramin (IC₅₀ = 4.1 µM). Finally, docking and molecular dynamics simulations provided the theoretical basis for the favorable activity of the designed compounds.     

寡聚（4-乙烯基苯基磷酸）/金纳米粒子的磷酸酶可视化检测研究

基于金属纳米粒子的磷酸酶可视化检测技术具有重要的生物医用前景. 然而, 文献中报道的方法大多采用多肽或核酸等生物分子作为磷酸酶感应基团. 由于这些生物分子在水溶液中不稳定, 且可被磷酸酶以外的生物酶类降解, 导致这些方法在实际应用中存在局限性. 首次合成了一种化学结构稳定的水溶性寡聚(4-乙烯基苯基磷酸)[oligo(4-vinyl-phenyl phosphate), OVPP], 并制备得到由OVPP稳定的磷酸酶响应型金纳米粒子Au@OVPP, 最后以酸性磷酸酶为例, 考察了Au@OVPP在磷酸酶可视化检测方面的应用. 由于OVPP的亲水性, Au@OVPP可在水溶液中稳定分散; 当体系中存在磷酸酶时, 由于OVPP中的亲水基团(磷酸根)被磷酸酶水解释放, 使得Au@OVPP的稳定分子OVPP由亲水变为疏水, 进而导致金纳米粒子的聚集及溶液宏观颜色的改变. 通过(肉眼)观察Au@OVPP溶液的变色及程度, 建立了一种灵敏、专一和低成本的磷酸酶检测方法. 该方法对于酸性磷酸酶的(肉眼)检测下限约为10 U/mL, 且对多种生物分子具有较好的抗干扰能力.     

高速逆流色谱制备分离紫甘薯花色苷

采用高速逆流色谱法分离纯化紫甘薯花色苷.以正丁醇-乙酸乙酯-0.5%乙酸(3:1:4,V/V)为溶剂体系,上相为固定相,下相为流动相,流速2mL/min,进样量300mg,分离得到两种花色苷的混合物;混合物再以0.2%三氟乙酸-正丁醇-甲基叔丁基醚-乙腈(6:5:2:1,V/V)为溶剂体系,上相为固定相,下相为流动相,...     

Bioreactors for H2 Production by Purple Nonsulfur Bacteria

Two types of laboratory-scale bioreactors were designed for H₂ production by purple nonsulfur bacteria. The bioreactors employed a unique type of hydrogenase activity found in some photosynthetic bacteria that functions in darkness to shift CO (and H₂O) into H₂ (and CO₂). The mass transport of gaseous CO into an aqueous bacterial suspension was the rate-limiting step and the main challenge for bioreactor design. Hollow-fiber and bubble-train bioreactors employing immobilized and free-living bacteria have proven effective for enhancing the mass transfer of CO. The hollow-fiber bioreactor was designed so that both a growth medium and CO (10% in N₂) passed from the inside of the fibers to the outside within the bioreactor. Bacteria were immobilized on the outer surface of the hollow fibers. Hydrogen production from CO at an average rate of 125 ml g cdw⁻¹ h⁻¹ (maximum rate of 700 ml g cdw⁻¹ h⁻¹) was observed for more than 8 months. The bubble-train bioreactor was built using polyvinyl chloride (PVC) tubing, wound helically on a vertical cylindrical supporting structure. Small bubbles containing CO were injected continuously through a needle/septum connection from the gas reservoir (20% CO). Up to 140 ml g cdw⁻¹ h⁻¹ of H₂ production activity was observed using this bioreactor for more than 10 days. Dr. Paul Weaver is deceased.     

Specific Differential Spectrophotdmetric Determination of Ascorbic Acid in Plants Using Ascorbate Oxidase

Abstract

A specific enzymic-differential spectrophatometric method is described and evaluated for the determination of ascorbic acid in vegetables and medicinal plants. It is based on the absorbance measurement at 593 nm of the complex of ferrous ion with 2,4,6-tris (pyridyl)-S-triazine, which is produced by reduction of ferric ion by ascorbic acid, versus a blank sample treated with ascorbate oxidase. The absorbance difference is linearly related to ascorbic acid from 10 to 100 ug/ml. A standard deviation of ±0.5 ug/ml (n=5) and a mean recovery of 101.47. (98.3–103.3%.) from spiked plant extracts, were found. The method was used to determine ascorbic acid in various plants of the Greek flora. Acacia cvanophvlla was found to be the richest source tested for ascorbic acid (89.6 mq/100 q of leaves). The method is very simple and can be used in routine analysis.     

Electrochemistry of ferric ruthenocyanide (Ruthenium Purple), and its electrocatalysis for proton reduction

Electrochemistry of ferric ruthenocyanide (Ruthenium Purple, RP) was studied in acidic solutions containing a sodium salt (NaH₂PO₄ or NaCl). Two kinds of redox peaks for the Fe^III/II couple were clearly seen in a Na⁺ solution, originating from a composite structure of both Fe₄^III[Ru^II(CN)₆]₃ (insoluble) and Fe^III[Ru^II(CN)₆]⁻ (soluble). In repeated CV scans in a NaCl solution, it was found that only the couple of the redox peak assigned to the insoluble form remains unchanged under the steady state. The ratio of the insoluble form was estimated by coulometry as ca. 40% of the initial coated unit cells of the RP. Efficient electrocatalytic H₂ formation was found to take place with the aid of the RP although this electrocatalysis was dependent on the type of sodium salt employed. In order to investigate the dominant factor affecting the overall kinetics in the H⁺ reduction catalysis, the dependences of both the catalytic activity and the electron transfer rate in the RP film were studied in a NaH₂PO₄ solution as a function of the coated amount. It was found that the overall kinetics are not dominated by electron transfer in the RP film.     

以2-磷酸抗坏血酸酯为底物检测碱性磷酸酯酶的微分脉冲伏安法

提出以2_磷酸抗坏血酸酯为碱性磷酸酯酶(ALP)底物微分脉冲伏安法测定ALP的方法 ;2_磷酸抗坏血酸酯在ALP的催化作用下发生水解反应生成抗坏血酸 ,抗坏血酸在玻碳电极上 +0.40V(vsAg/AgCl)被氧化而产生一个灵敏的氧化峰 ,氧化峰电流随着酶浓度的增大而增大 ,借助此氧化峰电流可以测定ALP ,进而可用于以ALP为标记酶的酶免疫分析 ;用微分脉冲伏安法对酶催化反应条件和酶催化反应产物的测定条件进行了详细的研究 ,建立了以2_磷酸抗坏血酸酯为底物的伏安酶联免疫分析新体系 ,测定游离ALP的线性范围是0.4～2.0×103 U/L,检测限为0.3U/L,对游离的IgG_ALP的测定最大稀释比为1∶200000。     

Xanthurenic Acid in the Shell Purple Patterns of Crassostrea gigas: First Evidence of an Ommochrome Metabolite in a Mollusk Shell

Ommochromes are one of the least studied groups of natural pigments, frequently confused with melanin and, so far, exclusively found in invertebrates such as cephalopods and butterflies. In this study focused on the purple color of the shells of a mollusk, Crassostrea gigas, the first evidence of a metabolite of ommochromes, xanthurenic acid (XA), was obtained by liquid chromatography combined with mass spectrometry (UPLC-MS). In addition to XA and various porphyrins previously identified, a second group of high molecular weight acid-soluble pigments (HMASP) has been identified with physicochemical and structural characteristics similar to those of ommochromes. In addition, fragmentation of HMASP by tandem mass spectrometry (MS/MS) has revealed a substructure common to XA and ommochromes of the ommatin type. Furthermore, the presence of melanins was excluded by the absence of characteristic by-products among the oxidation residues of HMASP. Altogether, these results show that the purple color of the shells of Crassostrea gigas is a complex association of porphyrins and ommochromes of potentially ommatin or ommin type.     

NLIP and HAD-like Domains of Pah1 and Lipin 1 Phosphatidate Phosphatases Are Essential for Their Catalytic Activities

Saccharomyces cerevisiae Pah1 phosphatidate phosphatase (PAP) catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol, controlling phospholipids and triacylglycerol metabolisms. Pah1 and human Lipin 1 are intrinsically disordered proteins with 56% and 43% unfolded regions, respectively. Truncation analysis of the conserved and non-conserved regions showed that N- and C-conserved regions are essential for the catalytic activity of Pah1. PAP activities can be detected in the conserved N-terminal Lipin (NLIP) domain and C-terminal Lipin (CLIP)/haloacid dehalogenase (HAD)-like domain of Pah1 and Lipin 1, suggesting that the evolutionarily conserved domains are essential for the catalytic activity. The removal of disordered hydrophilic regions drastically reduced the protein solubility of Pah1. Thioredoxin is an efficient fusion protein for production of soluble NLIP–HAD recombinant proteins in Escherichia coli.