Selective,switchable fluorescent probe for heparin based on aggregation-induced emission

A positively charged tetraphenylethene (TPE) derivative, TPE-4MN, was synthesized as a probe for heparin based on aggregation induced emission. On the addition of 5.0 μg/mL of heparin, TPE-4MN showed an enhanced emission of about 10-fold. The change in fluorescence at 475 nm was linear over a range of heparin concentrations of 0–1.0 μg/mL with an R = 0.99988 and the limit of detection (LOD) was calculated to be 0.75 μg/mL. The mechanism of the detection was proven to be through an ion pairing interaction. TPE-4MN showed good selectivity for heparin over other types of polysaccharides and could easily distinguish heparin from heparan sulfate, a glycosaminoglycan having a similar structure to that of heparin.     

A new approach for heparin standardization: combination of scanning UV spectroscopy, nuclear magnetic resonance and principal component analysis

The year 2007 was marked by widespread adverse clinical responses to heparin use, leading to a global recall of potentially affected heparin batches in 2008. Several analytical methods have since been developed to detect impurities in heparin preparations; however, many are costly and dependent on instrumentation with only limited accessibility. A method based on a simple UV-scanning assay, combined with principal component analysis (PCA), was developed to detect impurities, such as glycosaminoglycans, other complex polysaccharides and aromatic compounds, in heparin preparations. Results were confirmed by NMR spectroscopy. This approach provides an additional, sensitive tool to determine heparin purity and safety, even when NMR spectroscopy failed, requiring only standard laboratory equipment and computing facilities.     

平菇菌粗多糖的抗氧化活性研究

采用深层发酵技术生产平菇粗多糖,时其清除DPPH自由基、羟自由基的能力、铁离子螯合能力以及还原力进行了比较分析。结果表明：菌丝体粗多糖和发酵液粗多糖均具有较强的抗氧化能力,但2种多糖的抗氧化能力存在差异;茵丝体粗多糖清除DPPH自由基的能力较强,其EC。。值为2．20mg／mL;发酵液粗多糖清除羟自由基的能力、铁离子螯合能力以及还原力较强,其EC50值分别为0．72mg／mL、3．32mg／mL和7．91mg／mL。在一定的浓度范围内,多糖的浓度增加,其抗氧化能力也随之增强,呈量效依赖关系。     

High-performance liquid chromatographic separation of heparin-derived oligosaccharides

Heparin has been enzymatically depolymerized with heparinase (heparin lyase (EC 4.2.2.7)) and then separated into di-, tetra-, hexa-, octa-, and decasaccharide mixtures by low-pressure gel-permeation chromatography (GPC). These sized mixtures were resolved by strong anion-exchange (SAX) HPLC into multiple components. The fractions from the SAX-HPLC were collected and characterized for size by GPC-HPLC and sulfate content by ion chromatography. This study provides detailed methodology for the separation of larger and more highly sulfated oligosaccharides than previously reported. It describes the first use of ion chromatography for the accurate determination of the sulfate content of heparin oligosaccharides, a method which can also be applied to heparin and other glycosaminoglycans.     

A novel HPLC fluorescence method for the quantification of methylphenidate in human plasma

A number of analytical methods have been established to quantify methylphenidate (MPH). However, to date no HPLC methods are applicable to human pharmacokinetic studies without the use of mass spectrometry (MS) detection. We developed a sensitive and reliable HPLC-fluorescence method for the determination of MPH in human plasma using 4-(4,5-diphenyl-1H-imidazol-2-yl) benzoyl chloride (DIB-Cl) as the derivatizing agent. An established GC-MS method was adopted in this study as a comparator assay. MPH was derivatized using DIB-Cl, and separated isocratically on a C18 column using a HPLC system with fluorescence detection (lambda(ex)=330 nm, lambda(em)=460 nm). The lower limit of quantification was found to be 1 ng/mL. A linear calibration curve was obtained over the concentrations ranging from 1 ng/mL to 80 ng/mL (r=0.998). The relative standard deviations of intra-day and inter-day variations were 相似文献   

多壁碳纳米管固定化生物酶修饰电极检测杂色曲霉素的初步研究

色曲霉素（ST）是一种致癌致畸的真菌毒素,已报道的检测ST的技术有TLC、HPLC、ELISA以及毒素基因的PCR检测技术。初步探索了酶生物传感器三电极系统对ST的电化学分析。在实验中,应用多壁碳纳米管作为分子识别元件ADTZ的固定化基质和传感器的电子传递体构建了Au工作电极,对ST进行CV和DPV分析,结果表明：ST在-600mＶ位置有一明显的特征还原峰电位,线性检测范围是8.32×10^-5～6656×10^-5mg/mL,检测下限为8.32×10^-5mg/mL,响应时间10s,为进一步的研究打下良好的基础。     

Microanalysis of glycosaminoglycan-derived oligosaccharides labeled with a fluorophore 2-aminobenzamide by high-performance liquid chromatography: application to disaccharide composition analysis and exosequencing of oligosaccharides

A series of disaccharides derived from chondroitin sulfate and heparin/heparan sulfate were derivatized at their reducing ends with a fluorophore 2-aminobenzamide to develop a sensitive microanalytical method for glycosaminoglycans. The resulting labeled compounds derived from chondroitin sulfate or heparin/heparan sulfate were well-separated and quantified by HPLC equipped with a fluorescence detector. The detection limit was a low picomole level. This method was applied to the analysis of the disaccharide composition of tetra- and hexasaccharides derived from chondroitin sulfate and heparin/heparan sulfate as well as these glycosaminoglycan polysaccharides. The method was also successfully applied to the exosequencing of chondrohexasaccharides, where the fluorophore-labeled oligosaccharides were degraded exolytically from the nonreducing ends using bacterial eliminases. The resultant labeled fragments were identified by HPLC.     

Purification and characterization of heparin from the Italian clam Callista chione

An unusual heparin (approximately 1.9 mg/g of dry tissue) was isolated from the marine italian bivalve mollusk Callista chione. Agarose gel electrophoresis showed a high content of the fast-moving heparin component (85 +/- 7.6%) and 15 +/- 1.3% of the slow-moving species. An average molecular mass of 10 950 was calculated by PAGE analysis. The anticoagulant properties were measured as APTT (97 +/- 12.1 IU/mg) and anti-Xa activity (52 +/- 7.4 IU/mg). Structural analysis of clam heparin, performed by depolymerizing heparin samples with heparinase (EC 4.2.2.7) and then separating the resulting unsaturated oligosaccharides by SAX-HPLC, revealed the presence of low amounts of the trisulfated disaccharide [DeltaUA2S(1-->4)-alpha-d-GlcN2S6S] and a significant increase of the disaccharides bearing nonsulfated iduronic and glucuronic acids, [-->4)-alpha-l-IdoA(1-->4)-alpha-d-GlcNAc6S(1-->] and [-->4)-alpha-l-IdoA(1-->4)-alpha-d-GlcN2S6S(1-->], and [-->4)-beta-d-GlcA(1-->4)-alpha-d-GlcN2S6S(1-->]. As a consequence, Callista chione heparin is a low-sulfated polysaccharide showing a specific decrease of the sulfatation in position 2 of the uronic acid units.     

Dextran sulfate and heparin interact with CD4 molecules to inhibit the binding of coat protein (gp120) of HIV

Dextran sulfate, heparin, and certain other sulfated polysaccharides potently inhibit the adsorption of HIV to CD4+ cells. The mechanism of this inhibition is unclear and, specifically, it is unknown if these agents act at the level of CD4-gp120 binding. For example, previous reports have demonstrated that dextran sulfate does not inhibit the cell surface binding of anti-CD4 mAb known to be directed at the gp120 binding site. In order to confirm and extend these observations, in the present study, it was shown that dextran sulfate does not inhibit the binding of OKT4A, OKT4C, Leu3a, or B66.6 to CD4+ cells as measured by cytofluorography. Next, recombinant forms of CD4 (rT4) and gp120 (rgp120) were utilized to directly study their molecular interaction in the absence of other viral or cellular structures. Reciprocal solid phase ELISA assays were developed to study directly the effects of sulfated polysaccharides on the binding of rT4 to immobilized rgp120 and vice versa. Dextran sulfate, heparin, and fucoidan, but not chondroitin sulfate, inhibited the binding of rgp120 to rT4. Importantly, dextran sulfate and heparin pre-treatment of immobilized rT4, but not immobilized rgp120, inhibited rT4-rgp120 binding. Taken together, these data suggest that while both sulfated polysaccharides and anti-CD4 mAb inhibit gp120 binding, the sulfated polysaccharides interact with sites on CD4 that are distinct from those with which the antibodies bind.     

基于[Ru(phen)2(bpip)] 2+-DNA相互作用的共振光散射增强建立纳克级DNA的测定方法

通过研究钌多吡啶类配合物［Ru(phen)2(bpip)］2+与DNA相互作用的共振光散射等光谱,我们发现［Ru(phen)2(bpip)］²⁺与DNA相互作用的方式包括插入作用和静电作用模式.同时基于［Ru(phen)2(bpip)］²⁺ DNA体系增强的共振光散射现象,建立了一种简单、快速的测定纳克级核酸的新方法.实验结果表明体系在373 nm处共振光散射强度的增强与DNA的浓度呈线性关系.线性范围为0.025～1.250 mg/L,线性公式为△I_RLS=283.14C+2.26 (mg/L),相关系数为0.9983,DNA的检出限为5.7 ng/mL. 应用到实际样品的分析中,结果令人满意.     

Embryotropic effect of glycosaminoglycans and receptors in development of porcine pre-implantation embryos

The present study investigated the expression of receptors for glycosaminoglycans (GAGs) and the effect of GAGs supplementation on development of porcine IVF embryos. Total RNA was prepared from oocytes, 2-, 4- and 8-cell embryos, morulae and blastocysts. The expression of hyaluronic acid receptor (CD44) and heparin (HP) interacting protein (HIP) was determined using RT-PCR and Southern blot analysis. The CD44 and HIP mRNA were detected from in vitro matured oocytes and all stages of pre-implantation embryos. The IVF embryos were cultured in modified NCSU-23 medium supplemented with various concentrations (0, 0.1, 0.5 or 1.0 mg/mL) of hyaluronic acid (HA) or heparin. Supplementing with 0.5 mg/mL HA significantly increased total cell number compared to other experimental groups, due to increase in trophectoderm cells. Supplementing with 1.0 mg/mL, HP significantly increased blastocyst formation rate compared to the control group. Supplementing media, in which IVF embryos were cultured with 0.5 mg/mL HA + 1.0 mg/mL HP, significantly increased blastocyst formation rate compared to the control group. In conclusion, the present study demonstrated the expression of HA and HP receptors and the embryotrophic effect of HA or HP on porcine IVF embryos.     

Lymphocytosis produced by heparin and other sulphated polysaccharides in mice and rats

Lymphocytosis has been produced in mice and rats using heparin and other sulphated polysaccharides. Two hours after heparin (50 mg/kg ip) the concentration of lymphocytes in mouse blood increased threefold; it fell to control levels after 9 hr. The height of the lymphocytosis was related to the dose of heparin injected. After intravenous heparin in rats there was a comparable lymphocytosis maximal 1 hr after injection. In mice other negatively charged sulphated polysaccharides also caused lymphocytoses, which were greater and occurred later with increase in molecular weight of the substance injected. Results in rats were similar. No lymphocytosis followed the injection of negatively charged phosphated dextrans, positively charged DEAE dextran, or neutral dextran. There was no correlation between the effect of these substances on lymphocytes and their effect on coagulation of blood, hepatic phagocytosis, or the immune response to sheep red blood cells.     

Structural variation in the antithrombin III binding site region and its occurrence in heparin from different sources

A tetrasaccharide possessing a biosynthetically permissible structural variability in and adjacent to the antithrombin III (ATIII) binding site has been isolated from heparin lyase depolymerized bovine lung heparin by using strong anion-exchange high-pressure liquid chromatography (SAX-HPLC). On the basis of two-dimensional 500-MHz 1H NMR experiments, including phase-sensitive correlated spectroscopy (COSY) and rotating frame nuclear Overhauser enhancement spectroscopy (ROESY), and fast-atom bombardment mass spectrometry (FAB-MS), the primary structure of this tetrasaccharide was unambiguously established as delta UAp2S (1----4)-alpha-D-GlcNp2S6S(1----4)-beta-D-GlcAp(1----4)-alph a-D-GlcNp2S3S6S (where delta UA represents 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid). The 1H NMR ROESY experiment proved to be particularly valuable in offering sequence information. Heparins from a variety of species and tissue sources were examined by oligosaccharide mapping using SAX-HPLC and gradient polyacrylamide gel electrophoresis. Two of these heparins are used as anticoagulants; they are porcine intestinal mucosal heparin and bovine lung heparin. The predominant ATIII-binding site in porcine heparin contained an N-acetylated glucosamine residue. We now report the structure of the predominant ATIII-binding site in bovine heparin as----4)-alpha-D-GlcNp2S6S(1----4)-beta-D-GlcAp(1----4)-alph a-D- GlcNp2S3S6S(1----4)-alpha-L-IdoAp2S(1----4)-alpha-D-GlcNp 2S6S(1----. This study shows the presence of one or both types of ATIII-binding-site variants in all of the heparins that were examined.     

Chemical composition and bioactivities of a water-soluble polysaccharide from the endodermis of shaddock

The chemical composition of shaddock (Citrus paradisi) mainly consisted of polyphenols, proteins and polysaccharides. However, polysaccharides from shaddock materials have received much less consideration than polyphenols. Herein, a water-soluble neutral polysaccharide from the endodermis of shaddock was isolated and showed good bioactivities. Crude polysaccharides from the endodermis of shaddock (EPS) was extracted with hot water and separated on a DEAE Sepharose FF gel filtration column to obtain NEPS. The IR and UV spectra of NEPS showed that NEPS was mainly composed of polysaccharide and there are no proteins existing in NEPS. The DPPH radical scavenging and reducing power of NEPS are much lower than those of crude EPS; however, Citrus flavonoids significantly improved the DPPH radical scavenging potential and reducing power of NEPS. The crude EPS (5mg/mL) showed a similar inhibitory effect (77.92±5.03%) with NEPS (5mg/mL) (74.63±4.71%) on α-amylase.     

肝素纯化工艺中离子交换树脂的选择与应用研究

研究比较了5种树脂对肝素的吸附能力,从中选出S5428阴离子交换树脂来纯化肝素。通过对各因素的研究,确定了树脂对肝素的静态、动态吸附以及解吸的最佳条件。结果表明:静态吸附的温度45℃,pH 8.0的条件下吸附2 h,肝素的吸附率为90.5%;层析柱的动态吸附温度45℃,肝素溶液进样浓度1.0 mg/mL,进样速度1.5 mL/min,树脂柱能处理1.5 BV肝素液而不发生泄露,吸附量为3.05 mg/mL,达到饱和吸附时可处理4BV的料液,吸附量为9.18 mg/mL;采用2.0 mol/L NaCl洗脱,洗脱流速1.5 mL/min,肝素解吸率可达95.8%,肝素效价可达150 U/mg,效价回收率98%。     

血清游离精氨酸的快速检测

建立快速、准确的精氨酸定量检测方法。采用 6 30 0黄金系统氨基酸分析仪 ,在锂柱生理体液分析方法基础上 ,建立血清游离精氨酸 (ARG)快速测定方法。血清样本经磺基水杨酸沉淀蛋白后取上清液进行色谱分析 ,色谱柱为Beckman公司阳离子交换柱 (12cm× 4 .0mm) ;流动相为 2 0mmol·L- 1 柠檬酸锂水溶液 ,流速为 2 0ml·h- 1 ;比色波长 5 70nm。该法检测精氨酸浓度的线性范围为 5mg·L- 1 ～ 5 0mg·L- 1 ,相关系数 0 .99834,最低检测限 1mg·L- 1 ,重复性 :日内RSD 0 .4 0 % ,日间RSD 0 .5 5 % ,回收率 97.6 7%～ 10 0 .6 7% (平均值 99.0 7% ) ;整个实验过程耗时 2 8min。该法简便、快速、准确、可靠 ,适用于临床和科研工作。     

Label-free colorimetric sensor for ultrasensitive detection of heparin based on color quenching of gold nanorods by graphene oxide

A novel label-free colorimetric strategy was developed for ultrasensitive detection of heparin by using the super color quenching capacity of graphene oxide (GO). Hexadecyltrimethylammonium bromide (CTAB)-stabilized gold nanorods (AuNRs) could easily self-assembly onto the surface of GO through electrostatic interaction, resulting in decrease of the surface plasmon resonance (SPR) absorption and consequent color quenching change of the AuNRs from deep to light. Polycationic protamine was used as a medium for disturbing the electrostatic interaction between AuNRs and GO. The AuNRs were prevented from being adsorbed onto the surface of GO because of the stronger interaction between protamine and GO, showing a native color of the AuNRs. On the contrary, in the presence of heparin, which was more easily to combine with protamine, the AuNRs could self-assembly onto the surface of GO, resulting in the native color disappearing of AuNRs. As the concentration of heparin increased, the color of AuNRs would gradually fade until almost colorless. The amounts of self-assembly AuNRs were proportional to the concentration of heparin, and thereby the changes in the SPR absorption and color had been used to monitor heparin levels. Under optimized conditions, good linearity was obtained in a range of 0.02-0.28 μg/mL (R=0.9957), and a limit of detection was 5 ng/mL. The simultaneous possession of high sensitivity and selectivity, simplicity, rapidity, and visualization enabled this sensor to be potentially applicable for ultrasensitive and rapid on-site detection toward trace heparin.     

Collective sampling of intact anionic polysaccharide components and application in quantitative determination by LC-MS

Anionic polysaccharides, such as glycosaminoglycans (GAGs) and alginate, readily undergo source-induced fragmentation when analyzed by electrospray mass spectrometry with the use of high source cone voltage. The dissociation chemistry converts all components of a polysaccharide into a small set of structurally characteristic small saccharides. This chemistry enables the collective detection of a polysaccharide through the detection of one or more small saccharides. This ability, combined with the elution of polysaccharides as relatively compact bands using ion-pairing reverse phase liquid chromatography, created a unique opportunity for the development of LC–MS methods suitable for the quantitative analysis of intact anionic polysaccharides. Feasibility of this approach is demonstrated with a mixture of heparin, chondroitin sulfate A, and alginate.     

Comparison of colorimetric and chemiluminescent enzyme‐linked immunosorbent assay for the detection of endosulfan in food samples

Pesticides have become part of food protection since their inception. Endosulfan, an organochlorine insecticide, has been used against insect pests such as whiteflies, aphids, red spiders and mites. Methods of immunochemical assays have been devised for the determination and analysis of pesticides and commonly used for the analysis of contaminants in food, water, soil and body fluids. Chicken IgY antibodies raised against endosulfan haptens were used for the detection of endosulfan. We have compared colorimetric (CO) and chemiluminescence (CL) enzyme‐linked immunosorbent assay (ELISA) techniques for the detection of endosulfan isomers in a food matrix. CL ELISA assay was found to be more sensitive than CO assay. The mean recovery was 81.2–95.6% for α‐ and β‐endosulfan‐spiked food samples with 2.8–4.6% relative standard deviation. The detection of the endosulfan isomers was linear in the range 100 µg/mL–5 fg/mL, with a limit of detection at 100 µg/mL and 5 fg/mL for the CL ELISA method and 100 µg/mL and 1 ng/mL for the CO ELISA method respectively. These methods can be used for the rapid and reliable detection of organochlorine pesticide endosulfan. Copyright © 2015 John Wiley & Sons, Ltd.     

Semi-synthetic heparin derivatives: chemical modifications of heparin beyond chain length, sulfate substitution pattern and N-sulfo/N-acetyl groups

The glycosaminoglycan heparin is a polyanionic polysaccharide most recognized for its anticoagulant activity. Heparin binds to cationic regions in hundreds of prokaryotic and eukaryotic proteins, termed heparin-binding proteins. The endogenous ligand for many of these heparin-binding proteins is a structurally similar glycosaminoglycan, heparan sulfate (HS). Chemical and biosynthetic modifications of heparin and HS have been employed to discern specific sequences and charge-substitution patterns required for these polysaccharides to bind specific proteins, with the goal of understanding structural requirements for protein binding well enough to elucidate the function of the saccharide-protein interactions and/or to develop new or improved heparin-based pharmaceuticals. The most common modifications to heparin structure have been alteration of sulfate substitution patterns, carboxyl reduction, replacement N-sulfo groups with N-acetyl groups, and chain fragmentation. However, an accumulation of reports over the past 50 years describe semi-synthetic heparin derivatives obtained by incorporating aliphatic, aryl, and heteroaryl moieties into the heparin structure. A primary goal in many of these reports has been to identify heparin-derived structures as new or improved heparin-based therapeutics. Presented here is a perspective on the introduction of non-anionic structural motifs into heparin structure, with a focus on such modifications as a strategy to generate novel reduced-charge heparin-based bind-and-block antagonists of HS-protein interactions. The chemical methods employed to synthesize such derivatives, as well as other unique heparin conjugates, are reviewed.