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1.
Highly sensitive fluorescence detection of glycoprotein based on energy transfer between CuInS2 QDs and rhodamine B
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Xue Gao Dan Li Ying Tong Dan Ge Yiwei Tang Defu Zhang Jianrong Li 《Luminescence》2015,30(8):1389-1394
A highly sensitive fluorescence method for glycoprotein detection has been established based on fluorescence resonance energy transfer (FRET) between CuInS2 quantum dots (QDs) and rhodamine B (RB). Lectins comprise a group of proteins with unique affinities toward carbohydrate structures, so the process of FRET can occur between lectin‐coated QDs (CuInS2 QDs–Con A conjugates, acceptors) and carbohydrate‐coated RB (RB–NH2‐glu conjugates, donors). The fluorescence of lectin‐coated QDs was recovered in the presence of a glycoprotein such as glucose oxidase (GOx) and transferrin (TRF), which significantly reduced the FRET efficiency between the donor and the acceptor. Under optimal conditions, a linear correlation was established between the fluorescence intensity ratio I654/I577 and the TRF concentration over the range of 6.90 × 10‐10 to 3.45 × 10‐8 mol/L, with a detection limit of 2.5 × 10‐10 mol/L. The linear range for GOx is 3.35 × 10‐10 to 6.70 × 10‐8 mol/L, with a detection limit of 1.5 × 10‐10 mol/L. The proposed method was applied to the determination of glycoprotein in human serum and cell‐extract samples with satisfactory results. Furthermore, CuInS2 QDs–Con A conjugates are used as safe and efficient optical nanoprobes in HepG2 cell imaging. Copyright © 2015 John Wiley & Sons, Ltd. 相似文献
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3.
Hynek Mrázek Lenka WeignerováPavla Bojarová Petr NovákOndřej Vaněk Karel Bezouška 《Biotechnology advances》2013
The glycan code of glycoproteins can be conceptually defined at molecular level by the sequence of well characterized glycans attached to evolutionarily predetermined amino acids along the polypeptide chain. Functional consequences of protein glycosylation are numerous, and include a hierarchy of properties from general physicochemical characteristics such as solubility, stability and protection of the polypeptide from the environment up to specific glycan interactions. Definition of the glycan code for glycoproteins has been so far hampered by the lack of chemically defined glycoprotein glycoforms that proved to be extremely difficult to purify from natural sources, and the total chemical synthesis of which has been hitherto possible only for very small molecular species. This review summarizes the recent progress in chemical and chemoenzymatic synthesis of complex glycans and their protein conjugates. Progress in our understanding of the ways in which a particular glycoprotein glycoform gives rise to a unique set of functional properties is now having far reaching implications for the biotechnology of important glycodrugs such as therapeutical monoclonal antibodies, glycoprotein hormones, carbohydrate conjugates used for vaccination and other practically important protein–carbohydrate conjugates. 相似文献
4.
Anna V. Gudmundsdottir 《Carbohydrate research》2009,344(3):278-2676
Glycoconjugates can be readily formed by the condensation of a free-reducing terminus and a strong α-effect nucleophile, such as a hydrazide or a hydroxylamine. Further characterization of a series of glycoconjugates formed from xylose, glucose and N-acetylglucosamine, and either p-toluenesulfonyl hydrazide or an N-methylhydroxylamine, was carried out to gain insight into the optimal conditions for the formation of these useful conjugates, and their stability. Their apparent association constants (9-74 M−1) at pH 4.5; as well, as rate constants for hydrolysis, at pH 4.0, 5.0 and 6.0 (37 °C), were determined. The half-lives of the conjugates varied between 3 h and 300 days. All the compounds were increasingly stable as the pH approached neutrality. Conjugate hydrolysis rates mirrored those found for O-glycoside hydrolysis where conjugates formed from electron-rich monosaccharides hydrolyzed more rapidly. 相似文献
5.
Emanuele Ferrari Mireille Engelen Enrico Monzani Michela Sturini Stefania Girotto Luigi Bubacco Luigi Zecca Luigi Casella 《Journal of biological inorganic chemistry》2013,18(1):81-93
Elucidating the structure and biosynthesis of neuromelanin (NM) would be an important step towards understanding its putative role in the pathogenesis of Parkinson’s disease. A useful complement to studies aimed at unraveling the origin and properties of this essentially insoluble natural substance is the preparation of synthetic derivatives that resemble NM. With this aim in mind, water-soluble conjugates between dopamine-derived melanin and bovine serum albumin (BSA) were synthesized. Melanin–BSA adducts were prepared with both eumelanic oligomers obtained through the oxidative polymerization of dopamine and pheomelanic oligomers obtained under the same conditions from dopamine and cysteine. Iron ions were added during the synthesis to understand the interaction between the pigment and this metal ion, as the NM in neurons in several human brain regions contains significant amounts of iron. The structures of the conjugates were analyzed by 1H NMR spectroscopy and controlled proteolysis/MS experiments. The binding of iron(III) ions was evaluated by ICP analysis and EPR spectroscopy. The EPR signal from bound iron(III) indicated high-spin octahedral sites and, as also seen for NM, the signal is coupled to a signal from a radical associated with the melanic components of the conjugates. However, the intensity of the EPR signal from iron suggested a reduced fraction of the total iron, indicating that most of the iron is strongly coupled in clusters within the matrix. The amount of paramagnetic, mononuclear iron(III) was greater in the pheomelanin–BSA conjugates, suggesting that iron clustering is reduced in the sulfur-containing pigment. Thus, the melanin–BSA conjugates appear to be good models for the natural pigment. 相似文献
6.
Lu Ma Yuancai Chen Xude Wang Mingzhou Xiong Yuanyuan Sun Xiaoshu Zhang Yuqing Zhao 《Bioorganic & medicinal chemistry letters》2018,28(17):2920-2924
Gemcitabine (GEM) is widely used in clinical practice in the treatment of cancer and several other solid tumors. Nevertheless, the antitumor effect of GEM is partially prevented by some limitations including short half life, and lack of tumor localizing. Carboxymethyl glucan (CMG), a carboxymethylated derivative of β-(1-3)-glucan, shows biocompatibility and biodegradability as well as a potential anticarcinogenic effect. To enhance the antiproliferative activity of GEM, four water soluble conjugates of GEM bound to CMG via diverse amino acid linkers were designed and synthesized. 1H NMR, FT IR, elementary analysis and RP-HPLC chromatography were employed to verify the correct achievement of the conjugates. In vitro release study indicated that conjugates presented slower release in physiological buffer (pH 7.4) than acidic buffer (pH 5.5) mimicking the acidic tumor microenvironment. Moreover, A549, HeLa and Caco-2 cancer cell lines were used to evaluate the in vitro cytotoxicity of conjugates and the results showed that binding GEM to CMG significantly enhanced antiproliferative activity of GEM on A549 cells. Therefore, these conjugates may be potentially useful as a delivery vehicle in cancer therapy and worthy of further study on structure-activity relationship and antiproliferative activity in vitro and in vivo, especially for lung tumor. 相似文献
7.
In order to understand the role of the glycans in glycoproteins in solution, structural information obtained by NMR spectroscopy is obviously required. However, the assignment of the NMR signals from the glycans in larger glycoproteins is still difficult, mainly due to the lack of appropriate methods for the assignment of the resonances originating from the glycans. By using [U-13C6,2H7]glucose as a metabolic precursor, we have successfully prepared a glycoprotein whose glycan is uniformly labeled with 13C and partially with D at the sugar residues. The D to H exchange ratios at the C1-C6 positions of the sugar residues have been proven to provide useful information for the spectral assignments of the glycan in the glycoprotein. This is the first report on the residue-specific assignment of the anomeric resonances originating from a glycan attached to a glycoprotein by using the metabolic incorporation of hydrogen from the medium into a glycan labeled with [U-13C6,2H7]glucose. 相似文献
8.
Location of the Glycoprotein in the Membrane of Sindbis Virus 总被引:26,自引:0,他引:26
RICHARD W. COMPANS 《Nature: New biology》1971,229(4):114-116
SINDBIS virus, which is transmitted by arthropods, consists of a nucleoprotein core within a lipid-containing envelope. Its components assemble at a cellular membrane and virus particles form by an outfolding of this membrane. Thus, such viruses provide useful systems for studies of the structure and synthesis of membranes. The Sindbis virus particle contains only two proteins, one associated with the viral envelope and the other with the viral RNA in the core, or nucleocapsid1. The protein associated with the membrane is a glycoprotein, whereas the core protein contains no carbohydrate2. The exact location of the glycoprotein within the viral envelope has not been determined, nor has information been obtained about the function of the carbohydrate in the virion. The results described here indicate that the spikes which cover the surface of the virion are glycoprotein in nature. 相似文献
9.
Pilar Ruiz-Sánchez Christiane König Stefano Ferrari Roger Alberto 《Journal of biological inorganic chemistry》2011,16(1):33-44
It is attractive to use vitamin B12 as a carrier for targeted delivery of cytotoxic agents such as platinum complexes owing to the high demand for vitamin B12 by fast proliferating cells. The basic {B12–CN–PtII} conjugates are recognized by intracellular enzymes and converted to coenzyme B12 in an enzymatic adenosylation assay. The reductive adenosylation of {B12–CN–PtII} conjugates leads to the release of the PtII complexes; thus, {B12–CN–PtII} conjugates can be considered as prodrugs. It is important not only to elucidate the activity of the cisplatin–B12 conjugates, but also to understand the mode of action on a molecular level. Chemical reduction of {B12–CN–PtII} conjugates with cobaltocene yielded cob(II)alamin and induced release of the corresponding PtII species. Kurnakov tests and coordination of 2′-deoxyguanosine or GMP to the released PtII complexes allowed isolation and characterization of PtII complexes as released during enzymatic adenosylation. The biological activity of these PtII complexes was evaluated. Since the cleaved PtII complexes show cytotoxicity, the {B12–CN–PtII} conjugates can be used for specific targeting of cancer cells and therapeutic drug delivery. Preliminary in vitro cytotoxicity
studies indicated lower activity (IC50 between 8 and 88 μM) than found for pure cisplatin. Since active transport and receptor-mediated uptake limits the intracellular
{B12–CN–PtII} concentration, comparison with pure cisplatin is of limited use. We could show that the PtII complexes cleaved from B12 exerted a cytotoxicity comparable to that of cisplatin itself. Cytotoxicity studies in vitamin B12 free media showed a dependence on the addition of transcobalamin II for B12–Pt(II) conjugates. 相似文献
10.
MAC E. HADLEY FAHAD AL-OBEIDI VICTOR J. HRUBY JONATHAN C. WEINRACH DOUGLAS FREEDBERG JINWEN JIANG RACHEL S. STOVER 《Pigment cell & melanoma research》1991,4(4):180-185
Four fatty acids (FA, palmitic, myristic, decanoic, hexanoic) were individually conjugated to the N-terminus of the α-MSH fragment analog, H-Asp5-His6-D-Phe7-Arg8-Trp9-Lys10-NH2. This resulted in enhanced potency of the conjugates (compared to the unconjugated melanotropin analog) as determined in the lizard skin bioassay and in the mouse melanoma cell tyrosinase bioassay. The shorter conjugates of hexanoic and decanoic acid were at least equipotent to α-MSH in the lizard skin bioassay, whereas the longer myristoyl and palmitoyl analogs were 100 times less active. The myristoyl and palmitoyl conjugates exhibited a “creeping” potency in the lizard skin bioassay—that is, potency of the peptides increased with time in contact with the skins. These observations may be related to the more lipid nature of these FA-conjugates. In the tyrosinase assay, the conjugates were 10–100 times more active than α-MSH or the unconjugated analog. Each of the FA-melanotropic peptide conjugates exhibited prolonged (residual) melanotropic activity in both the lizard skin and melanoma cell bioassays. In other words, after removal of the melanotropin conjugates from contact with the skins or cells, responses were still manifested for hours or days thereafter. As little as 1 hr of contact with melanoma cells resulted in enhanced enzyme activity as measured 48 hr later. Since the conjugates, but not H-[Ast5,D-Phe7,Lys10]α-MSH5-10-NH2, exhibited prolonged activity, the conversion of reversible agonists to irreversible agonists was demonstrated. 相似文献
11.
Bovin NV 《Biochemical Society symposium》2002,(69):143-160
This chapter deals with the tendencies in design of multivalent neoglycoconjugates for glycobiology research and high-throughput profiling technologies, including cellular phenotyping. Soluble polyacrylamide (PAA) conjugates are remarkable owing to a variety of possibilities for synthesis and application. PAA is soluble and stable, and the molecule is flexible, PAA-tethered ligands are capable of adjusting to a receptor during multiple-point interactions and PAA does not bind to the cell surface. Synthesis provides unlimited diversity of the probe types (biotin, fluorescein, allyl, digoxygenin, 3H, radiolabelled I), glyco-particles, glyco-surfaces, multiarrays, immunogens etc. Several examples illustrate the most advanced applications. (i) Dynamic systems: the selectin ligands immobilized on the surface as sugar-PAA conjugates made the study of the kinetics of rolling in the model system possible. Carbohydrate ligands that are covalently attached to the chip as sugar-PAA conjugates are of use with the surface plasmon resonance method. (ii) Pseudoglycoprotein: some questions arise regarding the biologically active glycoproteins, e.g.: is a carbohydrate or peptide fragment responsible for the activity? We have proposed the approach that promotes to answer this and other questions. The pool of oligosaccharides that were spitted off a glycoprotein is attached to PAA resulting in a pseudoglycoprotein. (iii) Virtual (dynamic) glycotope: receptor-ligand recognition, such as that of P-selectin with its receptor P-selectin glycoprotein ligand 1, frequently involves molecular interactions at two distinct sites. Using P-selectin as a model, we developed an approach to discover novel ligands. PAA was synthesized with multiple ligands; a marked synergistic inhibitory effect was observed. 相似文献
12.
J. E. Richmond 《Protoplasma》1982,110(1):34-38
Summary Dimethyl sulphoxide at relatively low comentrations, 0.01 to 1 mM, enhanced the conjugation and cell-to-cell adhesion of complementary strains of matingTetrahymena thermophila. The time required to form stable conjugates was reduced by dimethyl sulphoxide. This chemical stimulated the uptake of glycine and glucosamine from the suspending media. Incorporation of 2-14C-glycine and 6-3H-D-glucosamine into protein and glycoprotein was enhanced in whole cells, surface membrane and cilia. Incorporation of glucosamine into the microsomal fraction was increased in the dimethyl sulphoxidetreated cells while there was little change in glycine incorporation. There were no detectable changes in glycine and glucosamine incorporation into the nuclear fractions isolated from conjugatingTetrahymena exposed to dimethyl sulphoxide. 相似文献
13.
Summary
125I-monoclonal IgG anti-gamma chain antibodies were conjugated to ferritin using glutaraldehyde as a bifunctional reagent. The molar ratio of IgG:ferritin:glutaraldehyde resulting in the highest yield was determined. Free IgG was separated from IgG bound to ferritin by sucrose density gradient ultracentrifugation; free ferritin was separated from antibody-ferritin conjugates by differential salt precipitation. The IgF:ferritin molar ratio of the resulting product was 11.4, contained over 90% ferritin-IgG monomers; 70–90% of the 125I activity bound immunospecifically to sepharose-IgG or aggregated human globulin (AHG). The product was used as an immunologic EM marker for AHG. Monoclonal antibody-ferritin conjugates prepared by this method should prove useful for quantitative ultrastructural analysis of surface antigens.Dr. Rudick is the recipient of Teacher-Investigator Development Award PHS 1 KO7 NS 00791-01 相似文献
14.
Gelii V. Ponomarev Maria N. Solovieva Nikita O. Dugin Maria G. Zavialova Arif R. Mehtiev Alexander Yu. Misharin Roman A. Novikov Yaroslav V. Tkachev Vladimir I. Popenko Vladimir P. Timofeev 《Bioorganic & medicinal chemistry》2013,21(17):5420-5427
The chemical synthesis of six lipophilic conjugates of chlorins was carried out, in which lipophilic fragment (either hexadecyl- or cholest-5-en-3β-yloxyethyl-) bound to 131-, 152-, 173-positions of macrocycle by formation of related carboxamides. Structure of synthesized conjugates was studied by spectral methods and molecular modeling. Lipophilic conjugates of chlorins, being mixed with egg yolk phosphatidyl choline, formed mixed micelles stable in aqueous media under physiological conditions. Mixed micelles of conjugates with phosphatidyl choline differing in stoichiometric compositions were prepared and characterized by absorption spectra, electron microscopy and laser scattering. These micelles were found to bind and internalized by human breast carcinoma MCF-7 cells. The presented data reveal that modification of macrocycle with lipophilic substituents, solubilization of obtained conjugates in aqueous medium as mixed micelles with phospholipids, and transfer of mixed micelles to cells is simple approach for targeting of chlorin derivatives, which apparently may be used in photodynamic therapy. 相似文献
15.
The model conjugates phycocyanin-allophycocyanin (C-PC-APC) and phycoerythrocyanin-phycocyanin-allophycocyanin (PEC-C-PC-APC)
were synthesized by using a heterobifunctional coupling reagent N-succinimidyl-3-(2-pyridyldithio)propionate. The rod-core
complex (αβ)6
PCLRC
27(αβ)3
APCLC
8.9 and phycobilisomes were separated from Anabaena variabilis. Energy transfer features for the conjugates and the complexes
were compared. The absorption and fluorescence emission spectra indicated that the linker-peptides mediate interaction of
phycobiliproteins and prompt energy transfer. The energy transfer in the conjugates was detected by fluorescence emission
spectra and confirmed by the addition of dithiothreitol. The conjugates may be used as models for studying the energy transfer
mechanism in phycobilisomes.
This revised version was published online in September 2006 with corrections to the Cover Date. 相似文献
16.
《Life sciences》1987,40(8):697-704
We are currently developing amino-substituted MPTP analogues as useful probes for understanding the mechanism of MPTP toxicity and Parkinson's disease. One analogue, 4′-amino MPTP, induces a loss of striatal dopamine and is thus a suitable substitute for MPTP. This probe will be used as a histologically fixable MPTP which can be used to answer detailed anatomical questions concerning the sites of MPTP, MPP+ uptake and storage. In addition, antibodies have been raised against MPTP and MPP+ in rabbits using diazo-linked bovine serum albumin conjugates. The antibodies have been characterized with regard to their recognition of relevant structural analogues using an enzymelinked immunoassay (ELISA) procedure. Antibodies to MPTP detected MPTP in mouse brain extracts derived from as little as 5 μg of tissue. The antibodies will be used for immunohistochemical localization of 4′-NH2-MPTP and 4′-NH2-MPP+ in brain, as well as probes for the screening of parkinsonian brain tissue for any MPTP- or MPP+-like materials which might exist. 相似文献
17.
Nicole Remy-Heintz Richard Magous Jean-Pierre Bali 《Biochemical and biophysical research communications》1980,93(4):1217-1224
The labelling of α1-acid glycoprotein (AGP) with (3H)-sodium borohydride was compared to the labelling with (125I)-sodium iodide by the chloramine T method in view to its use in a radioimmunoassay. The tritium labelling allowed to reach a high specific radioactivity similar to that obtained with iodide ((3H)-AGP: 29.8 mCi/mg; (125I)-AGP: 30.5 mCi/mg). Each mole of sialic acid residue of AGP contains one atom of tritium. The stability of (3H)-AGP was better than that of (125I)-AGP as indicated by its immunoreactivity as a function of time. Immunoreactivities and standard curves were similar for the two tracers but affinity of antiserum was higher for (125I)-AGP than for (3H)-AGP. Tritium labelling by (3H)-borohydride will be very useful for glycoprotein antigens which cannot be labelled with (125I)-iodide. 相似文献
18.
The chemical reaction between (±)-styrene oxide and N-acetylcysteine produces both positional isomers ( and ) as a mixture of diastereoisomers with a preference for the benzylic thioether isomer (2 : 1). Synthesis of the mercapturic acid conjugates from either (+)- or (?)-styrene oxide produces only two of the four possible stereoisomers. The single diastereoisomers of and were separated by high pressure liquid chromatography (HPLC) and identified by 1H- and 13C-nuclear magnetic resonance (NMR). The relative stereochemistry at the benzylic carbon center of the mercapturic acid conjugates was assigned on the basis of the established chemical correlation between optically pure styrene oxide and its precursor mandelic acid, and considerations on the mechanism of ring opening of epoxides by sulfur nucleophiles. The stereochemical definition of the isomers – should prove useful in investigations of the biotransformation of the glutathione (GSH) conjugates of styrene oxide. 相似文献
19.
Gabriella Sandri Gianluigi Sottocasa Enrico Panfili Gianfranco Liut 《生物化学与生物物理学报:生物膜》1979,558(2):214-220
Rat liver mitochondria may be subfractionated in sediment and supernatant fractions by swelling in the presence of EDTA and oxaloacetate. The sediment is largely depleted of the Ca2+-binding glycoprotein and its Ca2+-transporting activity may be as low as 10–20% of the starting value. Both the rate of Ca2+ uptake and the capacity to maintain a high Ca2+ concentration gradient across the membrane are depressed. Addition of an osmotic supernatant to the assay mixture may partially restore the original Ca2+-transporting ability. The active component in the supernatant is the Ca2+-binding glycoprotein. This is shown by the following facts: (a) the effect is enhanced by the addition of the purified glycoprotein to the supernatant; (b) precipitation of the glycoprotein from the supernatant by affinity chromatography-purified antibodies abolishes the stimulatory effect, and (c) in the presence of 130 μM Mg2+, the glycoprotein alone may restore fully the Ca2+-transporting ability of the particles. The maximal velocity is already reached at 0.1 μg glycoprotein/mg mitochondrial protein. 相似文献
20.
A method is described for preparing fluorescent conjugates of proteins labeled by reaction with methyl-C14-dansyl chloride and for determining their radioactivity by scintillation counting. The extinction coefficient of the bound dye varies in conjugates of different proteins and is considerably lower than the value generally employed to calculate degree of labeling. For assays of dansyl groups using absorption spectroscopy, a value of ? = 3.4 × 103M?1 cm?1 will probably be approximately correct. However, the radioactive dye technique allows much more accurate determinations of degree of labeling, as well as of the extinction coefficient, which may be of interest in itself. 相似文献