Engineering of Cardiotoxin by Chemical Synthesis: (I) Rapid Synthesis of Fully Active Cardiotoxin II and IV of Taiwan Cobra Venom

Two 60-residue snake toxins with four disulfide bridges, cardiotoxin II and IV, of Taiwan cobra (Naja naja atra) have been rapidly prepared in overall yields of 3.9% (cardiotoxin II) and 3.7% (cardiotoxin IV) within three weeks using the chemical method. Physicochemical characterization of these synthetic cardiotoxins was carried out by amino acid analysis, mass spectroscopy, capillary electrophoresis, peptide mapping, circular dichroism spectroscopy, and lethal toxicity. As compared with natural cardiotoxins, the results indicated that the synthetic cardiotoxins possessed the same physicochemical properties as those of natural ones. Therefore, in addition to preparation of various important toxins with satisfactory quantities for biochemical and pharmacological studies within a short lime, this rapid method also provides an important route to obtain many interesting toxins and designed toxin analogues for structure/function relationship studies in the near further.     

Effect of lubricants on the photo-oxidative degradation of solid poly(vinyl chloride)

Various commercially employed lubricants, such as low molecular weight polyethylene (Type PA-520 Wax), hydroxystearic acid (Loxiol G-21), butyl stearate (Loxiol G-32) and di-stearyl phthalate (Loxiol G-60), have been examined for their active rôle in the photo-oxidative degradation of poly(vinyl chloride) (PVC). The results show that low molecular weight polyethylene has an obvious accelerating effect on the photo-oxidative degradation of PVC, whereas other lubricants reduce the effect of photo-oxidation. None of the lubricants used has an obvious rôle in the chain scission and crosslinking of PVC.     

Alkyl Chain Length- and Polymorph-Dependent Photomechanochromic Fluorescence of Anthracene Photodimerization in Molecular Crystals: Role of the Lattice Stiffness

Anthracene–pentiptycene hybrid systems 1-Cn , where n refers to the number of carbon atoms in the linear alkyl chain, crystallize in three different polymorphs, denoted Y (yellow), G (green), and B (blue) forms in terms of the fluorescence color. While all Y-form crystals show the same yellow-to-blue fluorescence color response to the photomechanical stress generated by the anthracene [4+4] photodimerization reaction, the four G forms exhibit distinct photomechanofluorochromism (PMFC): from green to blue for G-1-C4 , to orange for G-1-C7 , to red for G-1-C8 , and to red then blue for G-1-C9 , and the B forms show no photochromic activity. The intriguing RGB three-color PMFC and abnormal topochemical reactivity of G-1-C9 are attributed to inherent softness of the crystal lattice.     

Synthesis of Taiwan Cobra Venom Cardiotoxin II Synthesis of Protected Hexapeptide,Sequence 44-49 and Protected Tripeptide,Sequence 50-52

The protected hexapeptide (sequence 44-49) and the protected tripeptide (sequence 50-52) of Taiwan Cobra (Naja naja atra) venom cardiotoxin were synthesized by stepwise coupling and fragment coupling methods. The protected hexapeptide is Nps-Lys(Z)-Ser-Ser-Leu-VaI-Leu-OMe and the protected tripeptides are Nps-Lys(Z)-Tyr(Bzl)-Val-OMe and Boc-Lys(Z)-Tyr(Bzl)-VaI-OMe.     

Identification and quantification of daidzein-7-glucuronide-4′-sulfate, genistein-7-glucuronide-4′-sulfate and genistein-4′,7-diglucuronide as major metabolites in human plasma after administration of kinako

Much attention has been paid to the metabolism and disposition of isoflavones daidzein (Dein) and genistein (Gein) with regard to the prevention of several hormone-dependent diseases. Recent studies have reported that several conjugates as well as aglycones may be biologically active or may be activated within target cells. However, the disposition of Dein and Gein in plasma is still uncertain. This paper describes the identification and quantification of the highly polar metabolites, daidzein-7-glucuronide-4′-sulfate (D-7G-4′S), genistein-7-glucuronide-4′-sulfate (G-7G-4′S), daidzein-4′,7-diglucuronide (D-4′,7-diG), and genistein-4′,7-diglucuronide (G-4′,7-diG) in human plasma after dietary administration of kinako (baked soybean powder) to two healthy volunteers. The structure identification of these conjugated metabolites in plasma was performed in comparison to the LC-ESI-MS and 600 MHz ¹H-NMR spectral data of the chemically synthesized compounds. Furthermore, 16 isoflavone metabolites including D-7G-4′S, G-7G-4′S, D-4′,7-diG, and G-4′,7-diG in plasma were simultaneously measured by a high-performance liquid chromatography–UV-diode-array detector method combined with solid-phase extraction using an Oasis HLB cartridge. D-7G-4′S, G-7G-4′S and G-4′,7-diG were found to be major metabolites of Dein and Gein in plasma, while intact aglycones were detected to be only ca. 2% in both subjects. The findings suggest that the conjugated metabolites could be the key compounds responsible for pharmacological and medicinal properties of isoflavones.     

Investigation of guanosine-quartet assemblies by vibrational and electronic circular dichroism spectroscopy, a novel approach for studying supramolecular entities

The self-assembly of guanosine-5'-hydrazide G-1 in D(2)O, in the presence and absence of sodium cations, has been investigated by chiroptical techniques: electronic (ECD) and the newly introduced vibrational (VCD) circular dichroism spectroscopy. Using a combination of ECD and VCD with other methods such as IR, electron microscopy, and electrospray ionization mass spectrometry (ESI-MS) it was found that G-1 produces long-range chiral aggregates consisting of G-quartets, (G-1)(4), subsequently stacked into columns, [(G-1)(4)](n), induced by binding of metal cations between the (G-1)(4) species. This process, accompanied by gelation of the sample, is highly efficient in the presence of an excess of sodium cations, leading to aggregates with strong quartet-quartet interaction. Thermally induced conformational changes and conformational stability of guanosine-5'-hydrazide assemblies were studied by chiroptical techniques and the melting temperature of the hydrogels formed was obtained. The temperature-dependent experiments indicate that the long-range supramolecular aggregates are dissociated by increasing temperature into less ordered species, monomers, or other intermediates in equilibrium, as indicated by MS experiments.     

Layer-by-layer assembly of zeolite crystals on glass with polyelectrolytes as ionic linkers

ZSM-5 crystals and glass plates tethered with trimethylpropylammonium iodide and sodium butyrate, respectively, (denoted as Z+, Z-, G+, and G-, respectively) were prepared. Treatment of G- with Z+ suspended in ethanol results in monolayer assembly of Z+ on G- (G-/Z+) with high surface coverage. The zeolite crystals have a strong tendency to closely pack and align with the b-axis normal to the glass plate, despite large positive zeta potentials. Subsequent treatment of G-/Z+ with Z- leads to second-layer assembly of Z- on G-/Z+ (G-/Z+/Z-), but with rather poor coverage. Sequential treatment of G+ with poly(sodium 4-styrenesulfonate)(Na+PSS-), poly(diallyldimethylammonium chloride) (PDDA+Cl-), and Na+PSS- followed by Z+ yields glass plates assembled with monolayers of Z+ with very high surface coverage through the composite polyelectrolyte linkers (G+/PSS-/PDDA+/PSS-/Z+). The zeolite crystals also have a strong tendency to closely pack and align with the b-axis perpendicular to the substrate plane. The binding strength between the zeolite crystals and glass plates is much higher in G+/PSS-/PDDA+/PSS-/Z+ than in G-/Z+. Repetition of the sequential PSS-/PDDA+/PSS-/Z+ layering for five cycles yields glass plates assembled with pentalayers of ZSM-5 crystals [G+/(PSS-/PDDA+/PSS-/Z+)(5)]. The observed degrees of coverage and alignment of zeolite crystals in each layer were very high up to the third layers despite the nonuniformity of the sizes and shapes of the zeolite crystals used in this study. This report thus demonstrates the feasibility of layer-by-layer assembly of micrometer-sized zeolite crystals on glass through electrostatic interaction between surface-bound, full-fledged ionic centers, especially by use of polyelectrolyes as the linkers.     

The effect of poly(ethylene glycol) on the activity and structure of glucose-6-phosphate dehydrogenase in solution

The effect of poly(ethylene glycol), PEG, on the enzymatic activity of glucose-6-phosphate dehydrogenase (G-6-PDH) in the oxidation of glucose-6-phosphate (G-6-P), using NADP+ as co-enzyme was investigated. The enzymatic activity was determined by means of spectrophotometry in three different media: pure Tris–HCl buffer, solution of PEG400 (20 wt.%) and of PEG4000 (20 wt.%), both in buffer. Comparing the enzymatic activity values measured in pure buffer with those in the polymer solutions, an increase in the enzymatic activity of 20% was observed in the presence of PEG400 as well as in PEG4000. Calorimetric studies indicated the absence of preferential interactions between G-6-PDH and PEG400 or PEG4000. Nevertheless, the interaction enthalpy, ΔH_int, between NADP+ and PEG400 and PEG4000 amounted to −9.3 and −26.7 kJ/mol, respectively. Small angle X-ray scattering (SAXS) measurements were performed in a higher concentration range. Data analysis performed from SAXS curves by means of the intra-particle distance distribution function p(r) and Guinier plots yielded for G-6-PDH in pure buffer and PEG400 solutions radius of gyration, R_g, of about 70 Å and in PEG4000 solutions, R_g of about 40 Å. The latter has the same dimension as that found in the dimeric crystallographic structure of G-6-PDH, evidencing that G-6-PDH preserves its dimeric configuration in PEG4000 solution. On the contrary, different aggregates of G-6-PDH are formed in the presence of either buffer or PEG400. These findings show that the presence of PEG in solution can exert an effect on the enzyme structure and activity.     

Das Verhalten verschiedener Fermentaktivit?ten der H?hnchenlinse w?hrend des ersten Lebensjahres

Metabolic data of the eye-lens show a strong dependence on the age of the investigated individuum. Not only the quantitative turn-over but also the qualitative order of reactions are affected. To get further informations about these age-depending processes the enzyme activities of lactate-dehydrogenase (LDH), malate-dehydrogenase (MDH), NAD-sorbitol-dehydrogenase (SDH), fructoaldolase (ALD), glucose-6-phosphate-dehydrogenase (G-6-PDH), glutamate-oxalacetic-transaminase (GOT) and glutamate-pyruvate-transaminase (GPT) were determined in lenses of chickens during the first year of life. The absolute activities decrease in the following ranking order LDH>MDH>ALD>GOT>G-6-PDH> SDH>GPT. For the performance of the enzyme activities in dependence of age and lens weight estimates were calculated by multiple, non-linear regression analysis. A matrix of coefficients of correlations is gamed, which allows extracting of 3 factors by factor analysis permitting the subdivision of the metabolism of the lens into partial scopes. Factor I represents the main tendency of evolution and is interpreted as metabolism of growth. It is loaded especially by the age, lens weight, activities of LDH, SDH and — with reversed sign — ALD. The main loading of factor II is given by the activities of MDH and GOT and it represents the metabolism of maintenance of physiological conditions. Factor III shows an adverse behaviour of G-6-PDH and GPT and is interpreted as metabolism of the superficial layers of the lens in account to its special anatomic-topographic situation. This interpretation could be assured by special experiments as the topographical distribution of the enzyme activities of G-6-PDH and GPT is adequate to the signs of the loadings of factor III. The activity of G-6-PDH mainly is located within the outer layers whereas the central lens nucleus is rich in GPT-activity.     

Protein Engineering III: Computer Aided Design of Disulfide-Bond Free Cobra Venom Cardiotoxin with a Novel Conformation and Biological Activity from Synthetic Peptides

Using the crystal structure of cobra venom cardiotoxin as a templet, a computer designed peptide with a novel conformation and biological activity has been synthesized chemically. The designed peptide utilized two calcium coordination sites instead of disulfide bridges to hold the conformation. The coordination sites were introduced at the cleft of three β-sheet strands by replacing the residues of Leu-1, Leu-26, Ser-28, Leu-48, and Ser-55 with Glu and using their γ-carboxyl groups as legends. The residues of Cys at positions 3, 14, 21, 38, 42, 53, 54, and 59 of the four disulfide bridges were changed with Gly to remove all the disulfide bonds. Circular dichroism spectra showed that the synthesized peptide has a conformation similar to that of the native cardiotoxin of a defined structure only in aqueous solutions with the presence of calcium ions. Immunoprecipitation assay, using the anti-cardiotoxin V, showed that in the presence of calcium ion the peptide had same cross reaction as that of native cardiotoxin. Hemolysis assay in the presence of calcium ion (150–250 mmol) and phospholipase A₂ showed that the peptide had 65–70% as much cytolytic activity as the native toxin.     

Shell click-crosslinked (SCC) nanoparticles: a new methodology for synthesis and orthogonal functionalization

A new methodology for the preparation of well-defined core-shell nanoparticles was developed, based upon the employment of a multifunctional crosslinker to coincidently stabilize supramolecular polymer assemblies and imbed into the shell unique chemical functionalities. Amphiphilic diblock copolymers of poly(acrylic acid)(80)-b-poly(styrene)(90) that had been assembled into micelles and partially functionalized throughout the corona with alkynyl groups were utilized as Click-readied nanoscaffolds for the formation of shell Click-crosslinked nanoparticles (SCCs). Divergently grown dendrimers of the zero, first, second, and third generations having increasing numbers of azide terminating groups ((N(3))(2)-[G-0], (N(3))(4)-[G-1], (N(3))(8)-[G-2], and (N(3))(16)-[G-3], respectively) were investigated as crosslinkers via Click reactions with the alkynyl groups to form covalent linkages throughout the block copolymer micelle corona, thus forming a crosslinked shell. The crosslinking reactions were characterized by (1)H NMR and IR spectroscopies, differential scanning calorimetry (DSC), and dynamic light scattering (DLS) measurements. Only the first generation dendrimer ((N(3))(4)-[G-1]) possessed a sufficient balance of polyvalency and water solubility to achieve crosslinking and establish a robust nanostructure. The resulting SCC was further characterized with atomic force microscopy (AFM), transmission electron microscopy (TEM), and analytical ultracentrifugation (AU). The dendritic crosslinker is important as it also allows for the incorporation of excess functionality that can undergo complementary reactions. Within the shell of this SCC the remaining azide termini of the dendrimer crosslinker were then consumed in a secondary Click reaction with an alkynyl-functionalized fluorescein to yield a fluorescently labeled SCC that was characterized with DLS, AFM, TEM, AU, UV-vis, and fluorescent measurements as a function of pH.     

Polymerization of styrenes carrying dendrons of the first,second and third generation

The synthesis of styrene monomers 5 (G-1,2,3) with Fréchet-type dendrons of the first, second and third generation, and their radically initiated polymerization to result in high molecular weight dendrimers 6 (G-1,2,3) are reported. First light-scattering investigations using 6 (G-2) show that this polymer is best described as a Gaussian chain.     

Purification and partial characterization of two lectin isoforms fromCratylia mollis mart. (camaratu bean)

Two additional electrophoretically distinct molecular forms, isoforms (iso) 2 and 3, with lectin properties were isolated fromCratylia mollis Mart, seeds (FABACEAE), by extraction with 0.15M NaCl and ammonium sulfate fractionation, followed by chromatography on Sephadex G-75 and Bio-Gel P-200 (iso 2), as well as CM-Cellulose and Sephadex G-75 (iso 3). Both isoforms were human group nonspecific and showed distinct specificity. Polyacrylamide gel electrophoresis resolved iso 2 and 3 in polypeptides of apparent mol wts 60 and 31 kDa, respectively; a distinct isoelectric focusing pattern was obtained for iso 2 and 3, under denaturing and reducing conditions.     

Chemiluminescence from polytetrafluoroethylene

The chemiluminescence emission from commercial samples of polytetrafluoroethylene, PTFE (Halon G-80 and Halon G-10), was studied at temperatures between 25 and 172°C. The emission intensity decreased on extended heating, removal of the surface layer, solvent extraction, or changing from an atmosphere of oxygen to an inert atmosphere. Films of PTFE deposited from the vapor phase showed comparatively little chemiluminescence under these conditions. Ozone-induced chemiluminescence was observed at 37°C from G-10 but not from G-80, or from films deposited from the vapor phase. The chemiluminescence intensity from ozone and G-10 was not reduced by solvent extraction.     

光响应性树枝状偶氮苯单分子层

偶氮苯树枝状分子通过中心羧基与金或者石英基体表面的氨基进行酰胺化反应得到树枝状分子单分子层修饰的表面,利用红外反射 吸收光谱（IRRAS）、UV-Vis和Raman光谱法研究了单分子层的结构和性能,利用椭偏谱法来测量四代树枝状（G-4）单分子层在不同异构化形态的分子层厚度,其中反式构象的单分子层厚度为（2.38±0.09） nm,顺式构象厚度为（1.88±0.08） nm。 利用表面等离子体共振（SPR）研究不同形态下G-4单层与不同等电点的蛋白质之间的相互作用关系,结果表明,G-4单层为负电位表面,能够吸附正电位的蛋白质,而且当树枝状单分子层形态从反式变为顺式时,表面电位变为更负,能吸附更多正电位的蛋白质。     

Recent progress in the electrochromatography of proteins

We have constructed a column for the application of an electrical field to a flowing packed bed of Chromatographic media (electrochromatography), and we have studied the effect the electrical field on the elution of bovine serum albumin, β-lactoglobulin A and B, α-lactalbumin, and myoglobin from packed beds of Sephadex beads. The elution behavior of the model proteins on Sephadex with a high degree of crosslinking (Sephadex G-25) and with a lower amount of crosslinking (Sephadex G-75) was measured. We confirmed that proteins exhibited an unexplained electrically driven retention on the column packed with G-75. The electrically driven retention effect was greatly reduced or absent in columns packed with the more highly crosslinked G-25. The potential of electrochromatography for high-resolution separations was shown by the partial separation of β-lactoglobulin A and B by using short columns with the electric field polarity such that electrophoresis opposed convective flow in the column. Certain commerical equipment, instruments or materials are identified in this article to specify adequately the experimental procedure. Such identification does not imply recommendation by NIST, nor does it imply that the materials or equipment are necessarily the best available for the purpose.     

眼镜蛇蛇油中甘油三酸酯的成分研究

用ＡｇＮＯ３－硅胶薄层板，氯仿＋丙酮（２０＋１）为展开剂，将蛇油中甘油三酸酯分离为５个组分，并用同样的色谱固定相和石油醚－氯仿－丙酮溶剂系统进行制备性分离。用质谱法对甘油三酸酯进行分子一级水平的化学研究，确定了眼镜蛇蛇油中５个主要的甘油三酸酯成分的结构形式为：１６∶０－１８∶０－１８∶０，１６∶０－１８∶０－１８∶１，１６∶０－１８∶２－１８∶１，１８∶２－１８∶２－１８∶０，１８∶２－１８∶２－１８∶１。     

Dynamic information for cardiotoxin protein desorption from a methyl-terminated self-assembled monolayer using steered molecular dynamics simulation

Dynamic information, such as force, structural change, interaction energy, and potential of mean force (PMF), about the desorption of a single cardiotoxin (CTX) protein from a methyl-terminated self-assembled monolayer (SAM) surface was investigated by means of steered molecular dynamics (SMD) simulations. The simulation results indicated that Loop I is the first loop to depart from the SAM surface, which is in good agreement with the results of the nuclear magnetic resonance spectroscopy experiment. The free energy landscape and the thermodynamic force of the CTX desorption process was represented by the PMF and by the derivative of PMF with respect to distance, respectively. By applying Jarzynski's equality, the PMF can be reconstructed from the SMD simulation. The PMFs, calculated by different estimators based upon Jarzynski's equality, were compared with the conventional umbrella sampling method. The best estimation was obtained by using the fluctuation-dissipation estimator with a pulling velocity of v = 0.25 nm/ns for the present study.     

Purfication of synthetic cardiotoxin by affinity chromatography

A polypeptide containing 60 amino acids with 4 disulphide bonds, synthesized by the solid-phase method, was highly purifed by anticardiotoxin-Sepharose affinity chromatography following gel filtration and CM-cellulose chromatography. The identification of the final product as cardiotoxin was confirmed by thin-layer chromatography on silica gel, polyacrylamide gel electrophoresis, amino acid analysis, circular dichroism spectra, N-terminal analysis and four biological tests.     

两种方法制备的磷酸铁锂/石墨烯复合材料的性能对比

以FeSO₄·7H₂O、NH₄H₂PO₄、H₂O₂、Li₂CO₃、C₆H₁₂O₆和自制的氧化石墨烯(GO)为原料,分别采用原位包覆法和非原位包覆法制备了石墨烯磷酸铁锂样品:LiFePO₄/C/G-1和LiFePO₄/C/G-2。用X射线衍射(XRD)、扫描电镜(SEM)、透射电镜(TEM)、交流阻抗(EIS)和充放电测试研究了两种包覆方法制备的样品的晶体结构、形貌和电化学性能。结果表明原位法包覆所得复合材料LiFePO₄/C/G-1具有更优秀的电性能:在2.5~4.1V充放电,0.1C和1C首次放电比容量分别为158.15和150.5mAh·g^-1,在1C倍率下循环500次后容量保持率达到98.3%。