• Title/Summary/Keyword: dichroism technique

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Drug-Biomacromolecule Interaction XII: Comparative binding study of sulfaethidole to bovine serum albumin by equilibrium dialysis, fluorescence probe technique, uv difference spectrophotometry and circular dichroism

  • Kim, Chong-Kook;Chun, Yang-Sook;Lah, Woon-Lyong
    • Archives of Pharmacal Research
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    • v.12 no.3
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    • pp.160-165
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    • 1989
  • Binding of sulfaethidole to bovine serum albumin was studied by equilibrium dialysis, fluorescence probe technique, uv difference spectrophotometry and circular dichroism. Equilibrium dialysis method enabled us to estimate the total number of drug binding sites of albumin molecule. For sulfaethidole, albumin had 6 primary and 40 secondary binding sites. The primary and secondary binding constants were 0.9 * 10/sup 5/ M/sup -1/ and 0.2 * 10/sup 6/ M/sup -1/, respectivitely. 1-Anilino-8-naphthalenesulfonate (ANS) and 2-(4-hydroxylbenzeneazo)- benzoic acid (HBAB) were used as the fluorescence probe and the uv spectrophotometric probe, respectively. In fluorescence probe technique, results indicated that the number of higher affinity drug binding site of albumin was 1 and the number of lower affinity drug binding sites of albumin was 3, and the primary and secondary drug binding constants for bovine serum albumin were 2.15 * 10/sup 5/M/sup -1/ and 1.04 * 10/sup 5/ M/sup -1/, respectively. In uv difference spectrophotometry, binding sites were 3 and binding constant was 1.88 * 10/sup 5/M/sup -1/. The above spectrophotometry, binding sites were 3 and binding constant was 1.88 * 10/sup 5/M/sup -1/. The above results suggest that several different methods should be used in ompensation for insufficient information about drug binding to albumin molecule given by only one method.

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Determination of Enantiopurity of Chiral Epoxides by Vibrational Circular Dichroism Spectroscopy (진동 원편광 이색성 분광기를 사용한 키랄 에폭사이드의 광학순도 분석)

  • Lee, Joo-Hyun;Lee, Choong-Young;Kim, Geon-Joong
    • Applied Chemistry for Engineering
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    • v.23 no.6
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    • pp.577-582
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    • 2012
  • In this work, vibrational circular dichroism (VCD) technique was applied for the determination of %EE of chiral compounds. It may provide an easy way to determine the %EE with a proper accuracy within 2% error ranges as well as the absolute configuration of enantiomers. We demonstrated herein a flow cell VCD (FT-VCD) technique for time-dependent %EE measurements. The simultaneous monitoring of the mole fraction and %EE for two chiral species (epichlorohydrin and glycidol mixture) in the mixture was shown to be successful without any further separation steps. Thus, we demonstrate that FT-VCD is an appropriate analytical tool to monitor the kinetics of reactions involving chiral molecules. FT-VCD also provides a convenient nondestructive approach for the time dependent determination of the optical purity of individual components in a reaction mixture containing chiral molecules.

Studies of Liquid Crystal Alignment on the Photosensitive Polyvinylfluorocinnamate (광감성 폴리비닐플루오로신나메이트의 액정 배향에 관한 연구)

  • Kim, Dong-Soo;Ahn, Won-Sool;Ha, Ki-Ryong;Buluy, O.;Reznikov, Yu.
    • Polymer(Korea)
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    • v.31 no.5
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    • pp.393-398
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    • 2007
  • We studied the mechanism of liquid crystal alignment on polyvinylfluorocinnamate (PVCN-F) films which were irradiated by UV using polarized fourier transform infrared (FT-IR) spectroscopy and ultraviolet/visible (UV/Vis) spectroscopy. UV irradiation of PVCN-F films caused decrease of vinylene -C=C- stretching peak area at $1638cm^{-1}$ and shift of conjugated C:0 stretching vibration at $1712cm^{-1}\;to\;1734cm^{-1}$ which is caused by nonconjugated C=O stretching nitration. To study the orientation direction of 5CB liquid crystal (LC) molecules in the liquid crystal cell with PUV irradiation, rubbing treatment or without any treatment on the PVCN-F alignment layer, we used polarized FT-IR dichroism technique. We successfully measured 5CB LC alignment directions, which are perpendicular to the irradiated PUV polarization direction and parallel to the rubbing direction in the liquid crystal cell without using dichroic dyes.

Simple reflection technique for measuring the electro-optic coefficient of NLO polymer (단순 반사 방법을 이용한 NLO polymer의 전기광학 계수 측정)

  • 지이권;이용산;조형준;윤국로;임종선;박광서
    • Proceedings of the Optical Society of Korea Conference
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    • 2000.02a
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    • pp.148-149
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    • 2000
  • 유기 고분자는 기존 무기물질의 한계성을 감안해 볼 때, 전기 및 광학적 특성이 우수하여 이에 대한 연구가 매우 활발히 진행되고 있다. 이중에서 Azo 화합물은 특별한 물리적, 광학적 성질과 그 잠재적인 응용성 때문에 계속해서 지금도 많은 관심을 끌고 있다. 그래서 Azo 화합물은 액정과 함께 비선형 광학성질을 가지는 물질에 응용될 수 있으며, 또한 광 유도현상의 이색성(dichroism)및 복굴절(birefringence) 현상 같은 특성을 나타내고 있다.$^{l)2)3)}$ 선형 광학 실험중에서 단순 입사 방법$^{4)}$ 을 이용하여 전기 광학 계수를 측정하였으며, 다시 확장 지수함수(stretched exponential)모델$^{5)}$ 로써 극화(poling)와 완화(relaxation)의 꼴을 보았다. (중략)

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Inclusion Complexation of Cyclodextrin with Prothionamide in Aqueous Solution

  • Kim, Shin-Tae;Kim, Shin-Keun
    • Journal of Pharmaceutical Investigation
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    • v.12 no.4
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    • pp.132-144
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    • 1982
  • The inclusion of ${\beta}-cyclodextrin$ $({\beta}-CyD)$ with prothionamide in aqueous phase was investigated by circular dichroism(CD), ultraviolet (UV) absorption, and solubility technique. The results suggested that a region of drug chromophore was located within the asymmetric center of ${\beta}-cyclodextrin$. Solubility and spectral changes were quantitatively treated to obtain stoichiometric ratio, which was found to be 1 : 1, and formation constants which were determined by solubility, CD, and UV method were 257, 367, and 389 $M^{-1}$, respectively. Also, the formation constant of the inclusion complex was determined by CD method at various pH. The result was that $K_c$ depended upon the pH of medium, and this fact also supported that thioamide moiety was accomodated in the cavity of ${\beta}-cyclodextrin$.

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Construction of Two Metal-ion Binding Sites to Improve the 3′-5′Exonuclease Activity of Taq DNA Polymerase

  • Park, Yong-Hyun;Kim, Jong-Moon;Choi, Hye-Ja;Kim, Seog-K.;Kim, Young-Soo
    • Journal of Microbiology and Biotechnology
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    • v.8 no.5
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    • pp.471-477
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    • 1998
  • Taq DNA polymerase from Thermus aquaticus is very useful in the polymerase chain reaction. Taq DNA polymerase is classified in the pol I family, represented by E. coli DNA polymerase I. The three-dimensional structural alignment of 3'-5'exonuclease domains from the pol I family DNA polymerases explains why Taq DNA polymerase does not carry out proofreading in polymerase chain reactions. Three sequence motifs, Exo I, II, and III, must exist to carry out 3'-5'exonuclease activity for proof- reading by a 3'-5'exonuclease reaction, but these are abolished in Taq DNA polymerase. The key catalytic module in 3'-5'exonuclease is two metal ions chelated by four active-site carboxylic amino acids. Taq DNA polymerase was mutagenized to construct the catalytic module in the active site. The circular dichroism technique supported the formation of the catalytic module, and the radioactive assay showed that the 3'-5'exonuclease activity doubled in the mutant Taq DNA polymerase.

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Room Temperature Ferromagnetism on Co and Fe Doped Multi-wall Carbon Nano-tube

  • Chae, K.H.;Gautam, S.;Yu, B.Y.;Song, J.H.;Augustine, S.;Kang, J.K.;Asokan, K.
    • Proceedings of the Korean Vacuum Society Conference
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    • 2011.02a
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    • pp.171-171
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    • 2011
  • Co and Fe doped multi-wall carbon nano-tubes (MWCNTs) synthesized by microwave plasma enhanced chemical vapor deposition (PECVD) technique are investigated with synchrotron radiations at Pohang Light Source (PAL) and European Synchrotron Radiation Facility (ESRF). Near edge x-ray absorption spectroscopy (NEXAFS) measurement at C K, Co $L_{3,2}$ and Fe $L_{3,2}$-edges, and x-ray magnetic circular dichroism (XMCD) at Co and Fe $L_{3,2}$-edges have been carried at 7B1 XAS KIST and 2A MS beamline, respectively, to understand the electronic structure and responsible magnetic interactions at room temperature. X-ray absorption spectroscopy (XAS) at C K-edge shows significant p-bonding and Co and Fe L-edges proves the presence of $Co^{2+}$ and $Fe^{2+}$ in octahedral symmetry. Co and Fe doped MWCNTs show good XMCD spectra at 300K. The effect on the magnetism is also studied through swift heavy ion (SHI) radiations and magnetism is found enhanced and change in the electronic structure in Co-CNTs is investigated.

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Investigation on the Effects of Three X${\rightarrow}$Histidine Replacements on Thermostability of ${\alpha}$-Amylase from Bacillus amyloliquefaciens

  • Haghani, Karimeh;Khajeh, Khosro;Naderi-Manesh, Hossein;Ranjbar, Bijan
    • Journal of Microbiology and Biotechnology
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    • v.22 no.5
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    • pp.592-599
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    • 2012
  • Bacillus licheniformis ${\alpha}$-amylase (BLA), a thermophilic counterpart of Bacillus amyloliquefaciens ${\alpha}$-amylase (BAA), is an appropriate model for the design of stabilizing mutations in BAA. BLA has 10 more histidines than BAA. Considering this prominent difference, in the present study, three out of these positions (I34, Q67, and P407; located in the thermostability determinant 1 region and Ca-III binding site of BAA) were replaced with histidine in BAA, using the site-directed mutagenesis technique. The results showed that the thermostability of P407H and Q67H mutants had increased, but no significant changes were observed in their kinetic parameters compared to that of the wild type. I34H replacement resulted in complete loss of enzyme activity. Moreover, fluorescence and circular dichroism data indicated a more rigid structure for the P407H variant compared with that of the wild-type BAA. However, the flexibility of Q67H and I34H mutants increased in comparison with that of wild-type enzyme.

Expressed Protein Ligation of 5-Enolpyruvylshikimate-3-phosphate (EPSP) Synthase: An Application to a Protein Expressed as an Inclusion Body

  • Kim, Hak-Jun;Shin, Hee-Jae;Kim, Hyun-Woo;Kang, Sung-Ho;Kim, Young-Tae
    • Bulletin of the Korean Chemical Society
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    • v.28 no.12
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    • pp.2303-2309
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    • 2007
  • Expressed protein ligation (EPL) technique, joining recombinantly expressed proteins to polypeptides, has been widely adopted for addressing various biological questions and for drug discovery. However, joining two recombinant proteins together is sometimes difficult when proteins are expressed insoluble and unrefoldable, because ligation-active proteins via intein-fusion are obtainable when they are folded correctly. We overcame this limitation coexpressing target protein with additional methionine aminopeptidase (MAP) which enhances removal of the initiation methionine of recombinantly expressed protein. Our approach demonstrated that two domains of 46 kDa 5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase, a target of herbicide glyphosate, were successfully joined by native chemical ligation, although its C-terminal domain was expressed as an inclusion body. The intein-fused N-terminal fragment of EPSP synthase (EPSPSN, residues 1-237) was expressed and the ligation-active thioester tagged N-terminal fragment (EPSPSN-thioester) was purified using a chitin affinity chromatography and mercapto-ethanesulphonate (MESNA) as intein thiolysis reagent. Its Cterminal fragment (EPSPSC, residues Met237-238CYS-427), expressed as an inclusion body, was prepared from an additional MAP-expressing strain. Protein ligation was initiated by mixing ~1 mM of EPSPSN-thioester with ~2 mM of EPSPSCCYS (residues 238CYS-427). Also we found that addition of 2% thiophenol increased the ligation efficiency via thiol exchange. The ligation efficiency was ~85%. The ligated full-length EPSP synthase was dissolved in 6 M GdHCl and refolded. Circular dichroism (CD) and enzyme activity assay of the purified protein showed that the ligated enzyme has distinct secondary structure and ~115% specific activity compared to those of wild-type EPSP synthase. This work demonstrates rare example of EPL between two recombinantly expressed proteins and also provides hands-on protein engineering protocol for large proteins.