• Title/Summary/Keyword: Covalent Bond

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Fusion Protein Cleavage by Urokinase Covalentley Immobilized to Activated Sepharose Gels (활성화된 Sepharose Gels에 공유결합으로 고정화된 Urokinase를 이용한 융합단백질 절단반응)

  • 서창우;강관엽;이효실;안상점;이은규
    • KSBB Journal
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    • v.15 no.1
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    • pp.42-48
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    • 2000
  • Urokinase (UK), a thrombolytic enzyme used to clear catheters obstructed by blood clots, can be also used industrially in the recombinant protein purification system to cleave a fusion protein linked with a certain fragment of GST. We have immobilized UK by covalent attachment to activated Sepharose 6B-Cl gels and evaluated its performance to cleave a fusion protein of hGH and GST. The Sepharose gels were activated by etherification with glycidol (2,3-epoxypropanol) and further oxidized with periodate resulting in glyceryl-Sepharose gels. After the activation treatment, surface density of the aldehyde groups was 7-30 $\mu$mol-aldehde/mL-gel. Immobilization yield was higher than 99% at high pH (10.5), and the immobilized UK maintained ca. 80% specific activity of the soluble UK. In a column reaction the cleavage yield heavily depended on the feed rate, and it was nearly 86% of that from soluble UK. And the immobilized UK was successfully regenerated by unfolding and refolding with 6M GuHCl. After cleavaging reaction, the monomeric hGH was purified by using expanded bed adsorption chromatography.

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Synthesis, Characterization and ESR Studies of New Copper(II) Complexes of Vicinal Oxime Ligands (Vicinal Oxime 리간드의 새로운 구리(II) 착물에 대한 합성, 특성 및 ESR 연구)

  • El-Tabl, Abdou S.;Shakdofa, Mohamad M.E.;El-Seidy, Ahmed M.A.
    • Journal of the Korean Chemical Society
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    • v.55 no.4
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    • pp.603-611
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    • 2011
  • Ethoxylacetyl oxime ligands [HL, (1) and $H_2L^1$, (3)] react with copper(II) acetate monohydrate yield octahedral and square planar complexes, respectively. The complexes have been postulated due to elemental analyses, IR, UVVis. spectra, magnetic susceptibility, conductivity and ESR spectra. Molar conductance of the complexes in DMF indicates a non-ionic character. The ESR spectra of [$(L)_2Cu(H_2O)_2$], (2) complex at room temperature and 77K are characteristic of an axial symmetry ($d_{x2-y2}$) with covalent bond character and have a large line width typical of dipolar interactions. However, [$(L^1)Cu$], (4) complex in the solid state showed spectra of marked broadening and loss of hyperfine splitting confirming spinexchange interactions between the copper(II) sites. The spectrum of the doped copper(II) complex at room temperature showed super-hyperfine splitting from coordinated nitrogen atoms and it has an axial type ($d_{x2-y2}$) with covalent bond character and an essentially square-planar arrangement around the copper(II) ion. The spectrum of [$(L^1)Cu$], (4) in frozen methanol at 77K was characteristic of the triplet state of a dimer species and the distance found between the two copper(II) centers was calculated and is equal to 4.8 ${\AA}$.

Investigation of the Copper (Cu) Binding Site on the Amyloid beta 1-16 (Aβ16) Monomer and Dimer Using Collision-induced Dissociation with Electrospray Ionization Tandem Mass Spectrometry

  • Ji Won Jang;Jin Yeong Lim;Seo Yeon Kim;Jin Se Kim;Ho-Tae Kim
    • Mass Spectrometry Letters
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    • v.14 no.4
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    • pp.153-159
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    • 2023
  • The copper ion, Cu(II), binding sites for amyloid fragment Aβ1-16 (=Aβ16 ) were investigated to explain the biological activity difference in the Aβ16 aggregation process. The [M+Cu+(z-2)H]z+ (z = 2, 3 and 4, M = Aβ16 monomer) and [D+Cu+(z-2)H]z+ (z = 3 and 5, D = Aβ16 dimer) structures were investigated using electrospray ionization (ESI) mass spectrometry (MS) and tandem mass spectrometry (MS/MS). Fragment ions of the [M+Cu+(z-2)H]z+ and [D+Cu+(z-2)H]z+ complexes were observed using collision-induced dissociation MS/MS. Three different fragmentation patterns (fragment "a", "b", and "y" ion series) were observed in the MS/MS spectrum of the (Aβ16 monomer or dimer-Cu) complex, with the "b" and "y" ion series regularly observed. The "a" ion series was not observed in the MS/MS spectrum of the [M+Cu+2H]4+ complex. In the non-covalent bond dissociation process, the [D+Cu+3H]5+ complex separated into three components ([M+Cu+H]3+, M3+, and M2+), and the [M+Cu]2+ subunit was not observed. The {M + fragment ion of [M+Cu+H]3+} fragmentation pattern was observed during the covalent bond dissociation of the [D+Cu +3H]5+ complex. The {M + [M+Cu+H]3+} complex geometry was assumed to be stable in the [D+Cu+3H]5+ complex. The {M + fragment ion of [M+Cu]2+} fragmentation pattern was also observed in the MS/MS spectrum of the [D+Cu+H]3+ complex. The {M + [y9+Cu]1+} fragment ion was the characteristic fragment ion. The [D+Cu+H]3+ and [D+Cu+3H]5+ complexes were likely to form a monomer-monomer-Cu (M-M-Cu) structure instead of a monomer-Cu-monomer (M-Cu-M) structure.

Theoretical Studies on Electronic Structure and Absorption Spectrum of Prototypical Technetium-Diphosphonate Complex 99mTc-MDP

  • Qiu, Ling;Lin, Jian-Guo;Gong, Xue-Dong;Ju, Xue-Hai;Luo, Shi-Neng
    • Bulletin of the Korean Chemical Society
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    • v.32 no.7
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    • pp.2358-2368
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    • 2011
  • Density functional theory (DFT) and time-dependent density functional theory (TDDFT) calculations, employing the B3LYP method and the LANL2DZ, 6-31G$^*$(LANL2DZ for Tc), 6-31G$^*$(cc-pVDZ-pp for Tc) and DGDZVP basis sets, have been performed to investigate the electronic structures and absorption spectra of the technetium-99m-labeled methylenediphosphonate ($^{99m}Tc$-MDP) complex of the simplest diphosphonate ligand. The bonding situations and natural bond orbital compositions were studied by the Mulliken population analysis (MPA) and natural bond orbital (NBO) analysis. The results indicate that the ${\sigma}$ and ${\pi}$ contributions to the Tc-O bonds are strongly polarized towards the oxygen atoms and the ionic contribution to the Tc-O bonding is larger than the covalent contribution. The electronic transitions investigated by TDDFT calculations and molecular orbital analyses show that the origin of all absorption bands is ascribed to the ligand-to-metal charge transfer (LMCT) character. The solvent effect on the electronic structures and absorption spectra has also been studied by performing DFT and TDDFT calculations at the B3LYP/6-31G$^*$(cc-pVDZ-pp for Tc) level with the integral equation formalism polarized continuum model (IEFPCM) in different media. It is found that the absorption spectra display blue shift in different extents with the increase of solvent polarity.

Influence of Surface Morphology and Substrate on Thermal Stability and Desorption Behavior of Octanethiol Self-Assembled Monolayers

  • Ito, Eisuke;Gang, Hun-Gu;Ito, Hiromi;Hara, Masahiko;No, Jae-Geun
    • Proceedings of the Korean Vacuum Society Conference
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    • 2012.08a
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    • pp.219-219
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    • 2012
  • The formation and thermal desorption behaviors of octanethiol (OT) SAMs on single crystalline Au (111) and polycrystalline Au, Ag, and Cu substrates were examined by X-ray photoelectron microscopy (XPS), thermal desorption spectroscopy (TDS), and contact angle (CA) measurements. XPS and CA measurements revealed that the adsorption of octanethiol (OT) molecules on these metals led to the formation of chemisorbed self-assembled monolayers (SAMs). Three main desorption fragments for dioctyl disulfide (C8SSC8+, dimer), octanethiolate (C8S+), and octanethiol (C8SH+) were monitored using TDS to understand the effects of surface morphology and the nature of metal substrates on the thermal desorption behavior of alkanethiols. TDS measurements showed that a sharp dimer peak with a very strong intensity on single crystalline Au (111) surface was dominantly observed at 370 K, whereas a broad peak on the polycrystalline Au surface was observed at 405 K. On the other hand, desorption behaviors of octanethiolates and octanethiols were quite similar. We concluded that substrate morphology strongly affects the dimerization process of alkanethiolates on Au surfaces. We also found that desorption intensity of the dimer is in the order of Au>>Ag>Cu, suggesting that the dimerization process occurs efficiently when the sulfur-metal bond has a more covalent character (Au) rather than an ionic character (Ag and Cu).

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Preparation and Characterizations of C60/Polystyrene Composite Particle Containing Pristine C60 Clusters

  • Kim, Jung-Woon;Kim, Kun-Ji;Park, Soo-Yeon;Jeong, Kwang-Un;Lee, Myong-Hoon
    • Bulletin of the Korean Chemical Society
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    • v.33 no.9
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    • pp.2966-2970
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    • 2012
  • Fullerene/polystyrene ($C_{60}$/PS) nano particle was prepared by using emulsion polymerization. Styrene and fullerene were emulsified in aqueous media in the presence of poly(N-vinyl pyridine) as an emulsion stabilizer, and polymerization was initiated by water soluble radical initiator, potassium persulfate. The obtained nano particles have an average diameter in the range of 400-500 nm. The fullerene contents in the nano particle can be controlled up to 15 wt % by varying the feed ratio, which was confirmed by themogravimetric analysis (TGA) and elemental analysis (EA). The structure and morphologies of the $C_{60}$/PS nano particles were examined by various analytical techniques such as dynamic light scattering (DLS), scanning electron microscope (SEM), transmission electron microscope (TEM), electron diffraction (ED) pattern, X-ray powder diffraction (XRD), and UV spectroscopy. Unlike conventional $C_{60}$/PS particles initiated by organic free radical initiators, in which the fullerene is copolymerized forming a covalent bond with styrene monomer, the prepared $C_{60}$/PS nano particles contain pristine fullerene as secondary particles homogeneously distributed in the polystyrene matrix.

Dyeing Properties of Nylon Fabric with Reactive Dyes Having Different Reactive Group

  • Lee, Byung-Sun;Lee, Jin-Ah;Lee, Kwon-Sun;Park, Jong-Ho;Kim, Sung-Dong
    • Textile Coloration and Finishing
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    • v.18 no.5 s.90
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    • pp.1-7
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    • 2006
  • The dyeing properties of the several reactive dyes possessing different types of the reactive group on nylon 6 fabric have been investigated. Dyeing of nylon fiber with commercial reactive dyes was dependent on the dye bath pH. As pH increased, the extent of exhaustion decreased due to the deprotonation of the amino end group. But, the extent of fixation increased because of the enhanced reactivity with nylon fiber. It was found that all reactive dyes could be applied to nylon 6 reasonably at pH 5-6. But, the conflicting requirements of dye exhaustion and covalent bond formation limited the extent of fixation efficiency. The best method to improve the extent of the fixation efficiency of the reactive dyes was the conversion of sulphatoethylsulphone group to vinylsulphone group before dyeing with trisodium phosphate. The converted Supra type reactive dye gave the highest extent of fixation efficiency, which was about 90%.

Immobilization of Endo- and Exoinulinase on Vinylsulfone Activated Agarose (Vinylsulfone Activated Agarose 에 Endo- 및 Exoinulinase의 고정화)

  • 한상배;송근섭;정용섭;손희숙;우순자;엄태봉
    • Microbiology and Biotechnology Letters
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    • v.20 no.1
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    • pp.20-24
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    • 1992
  • In order to reuse inulinase effectively, a method for immobilizing both endo- and exoinulinase to vinylsulfone activated agarose via covalent bond was investigated. The immobilized enzyme preparation had, respectively, 400 U for exoinulinase activity and 80 U for endoinu- Iinase activity per gram gel. A thermal stability by immobilization had increased in the case of exoinulinase. Optimum pHs for two immobilized enzymes were 4.4 to 5.0. Synergistic effect which depends on mixed ratio of two immobilized enzymes was the best when the mixed ratio of endo/exo lay between 0.1 and 0.5, and its activity of the mixed enzyme increased 1.7 times as compared to that of each immobilized enzyme. Inulinase activities of both of the immobilized enzymes did not change during 20 times experimental runs in a batch reactor.

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Electrochemical Determination of Immobilization Technique for Glucose Sensor Fabrication (포도당 센서의 제작을 위한 고정화 방법의 전기화학적 결정)

  • 정태훈;홍석인;노봉수;정용섭;윤정원;김태진
    • KSBB Journal
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    • v.13 no.1
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    • pp.52-57
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    • 1998
  • The present work proposes a simple electrochemical method applicable to any immobilization processes of oxidase using a Clark type oxygen electrode as a base transducer. The present work suggests an optimal immobilization technique among three different methods of glucose oxidase(GOD) onto one side of $37[\mu}$mthick blend membranes, composed o 80% of cellulose triacetate and 20% of polycaprolactone, on the basis of the maximum Michaelis-Menten parameter(Vm) determined by either steady state or transient analyses. The electrode system was made of disk type gold cathode(4mm diameter) and Ag/AgCl anode. One side of the blend membrane was in contact with the cathode surface while the other side was immobilized with GOD either in covalent-bond or cross-linked forms, the latter being covered by $25{\mu}$m thick dialysis membrane of cellulose acetate. The resultant current density was on-line monitored by a potentiostat while glucose level was varied from 1 to 20 mM. The present study shows that direct cross-linking of GOD with glutaraldehyde was mostly preferred for fabrication of glucose sensor, on the basis of resultant kinetic parameters from either steady state or transient analyses.

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Simultaneous Synthesis and Sintering of Titanium Carbide by HPCS(High Pressure-Self Combustion Sintering) (고압연소 소결(HPCS)법에 의한 탄화티타늄(TiC)의 합성 및 소결)

  • 김지헌;최상욱;조원승;조동수;오장환
    • Journal of the Korean Ceramic Society
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    • v.34 no.5
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    • pp.473-482
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    • 1997
  • Titanium carbide(TiC) has a poor sinterability due to the strong covalent bond. Thus, it is generally fabricated by either hot pressing or pressureless-sintering at elevated temperature by the addition of sintering aids such as nickel(Ni), molybdenum(Mo) and cobalt(Co). However, these sintering methods have the following disadvantages; (1) the complicated process, (2) the high energy consumption, and (3) the possibility of leaving inevitable impurities in the product, etc. In order to reduce above disadvantages, we investigated the optimum conditions under which dense titanium carbide bodies could be synthesized and sintered simultaneously by high pressure self-combustion sintering(HPCS) method. This method makes good use of the explosive high energy from spontaneous exothermic reaction between titanium and carbon. The optimum conditions for the nearly full-densification were as follows; (1) The densification of sintered body becomes high by increasing the pressing pressure from 400kgf/$\textrm{cm}^2$ upto 1200 kgf/$\textrm{cm}^2$. (2) Instead of adding the coarse graphite or activated carbon, the fine particles of carbon black should be added as a carbon source. (3) The optimum molar ratio of carbon to titanium (C/Ti) was unity. In reality, titanium carbide body which were prepared under optimum conditions had relatively dense textures with the apparent porosity of 0.5% and the relative density of 98%.

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