Contribution of individual zinc ligands to metal binding and peptide folding of zinc finger peptides

Little is known about the contribution of individual zinc-ligating amino acid residues for coupling between zinc binding and protein folding in zinc finger domains. To understand such roles of each zinc ligand, four zinc finger mutant peptides corresponding to the second zinc finger domain of Sp1 were synthesized. In the mutant peptides, glycine was substituted for one of four zinc ligands. Their metal binding and folding properties were spectroscopically characterized and compared to those of the native zinc finger peptide. In particular, the electronic charge-transfer and d-d bands of the Co(II)-substituted peptide complexes were used to examine the metal coordination number and geometry. Fluorescence emission studies revealed that the mutant peptides are capable of binding zinc despite removing one ligand. Circular dichroism results clearly showed the induction of an alpha-helix by zinc binding. In addition, the structures of certain mutant zinc finger peptides were simulated by molecular dynamics calculation. The information indicates that His23 and the hydrophobic core formed between the alpha-helix and the beta-sheet play an essential role in alpha-helix induction. This report demonstrates that each ligand does not contribute equally to alpha-helix formation and coordination geometry in the zinc finger peptide.     

Structural change of 1-butyl-3-methylimidazolium tetrafluoroborate + water mixtures studied by infrared vibrational spectroscopy

Mixtures of ionic liquid (IL, 1-butyl-3-methylimidazolium tetrafluoroborate, [BMIM][BF4]) and water with varying concentrations were studied by attenuated total reflection infrared absorption and Raman spectroscopy. Changes in the peak intensities and peak positions of CHx (x = 1, 2, 3) vibration modes of the cation of the IL and OH vibration modes of the water molecules were investigated. Peaks from normal-mode stretch vibrations of CH bonds belonging to the imidazolium ring of the cation did not change their positions, while those from the terminal methyl group of the butyl chain blueshifted by approximately 10 cm-1 with the addition of water. On the other hand, change in the spectral shape in the OH stretch vibration region shows hydrogen-bonding network of water molecules breaking down rapidly as the IL is added. Trends in the change of the peak positions and the peak intensities suggested qualitative change of the intermolecular structure in the [BMIM][BF4] + H2O mixture at 32 +/- 2 and 45 +/- 2 mol/L of water concentration.     

Analyses of the EPR responses of sucrose and L-alpha-alanine radicals induced by C, Ne, and Ar ion irradiations

Using electron paramagnetic resonance (EPR), we investigated the signal responses of sucrose and L-alpha-alanine radicals produced by heavy-ion irradiation with various linear energy transfers (LETs) and absorbed dose. The spin yields for the two compounds showed a linear relation with the absorbed dose, as well as a logarithmic correlation with the LET. A quantitative EPR analysis showed that sucrose was more sensitive to the particle species than that of alanine. Hence, the present EPR results imply that sucrose can be useful as a radiation indicator. Further analysis was carried out for the radical-production cross section, which showed that stable radicals of the two compounds were produced through collisions of several particles with a single molecule.     

Simultaneous Determination of Imidazole Amino Acids, Aromatic Amino Acids, and Creatinine by Dual-Mode Gradient HPLC Using a Low-Capacity Sulfo-Functionalized Poly(Ethylstyrene-Divinylbenzene) Resin Column

This paper presents a low-capacity cation-exchange chromatography method for the analysis of UV-absorbing dipeptides and amino acids. A newly marketed low-capacity cation-exchange column packed with sulfo-functionalized highly cross-linked macroreticular poly(ethylstyrene-divinylbenzene) copolymer was used for the simultaneous determination of imidazole amino acids, aromatic amino acids, and creatinine in urine samples. A dual-mode binary gradient chromatography method was established using two solvents, A: 15 mM H₃PO₄/5 mM ethylenediamine and B: 15 mM H₃PO₄/5 mM ethylenediamine/40 (v/v) % CH₃CN at 40 °C, with an optimized time program for changing the delivery ratio of A/B and the flow rate. Good chromatograms were obtained within an acceptable cycle time of 25 min. The quantification data were satisfactory for all analytes, showing the relative standard deviations (RSD) of retention times between 0.08 and 1.68 %; RSDs of area intensities between 0.23 and 2.60 %; and linear regression lines with r ² more than 0.9994. The method could determine the creatinine ratios of the diagnostic markers on the single chromatographic run, which enabled to discriminate disease from health. For example, the creatinine ratios for phenylketonuria were significantly higher than those for controls. The method can provide highly cost-efficient information or useful knowledge for clinical and pharmaceutical studies.     

Micropore-free surface-activated carbon for the analysis of polychlorinated dibenzo-p-dioxins-dibenzofurans and non-ortho-substituted polychlorinated biphenyls in environmental samples

2,3,7,8-Substituted polychlorinated dibenzo-p-dioxins/polychlorinated dibenzofurans (PCDD/Fs) and non-ortho-substituted polychlorinated biphenyls (PCBs) account for almost all of the total toxic equivalents (TEQ) in environmental samples. Activated carbon columns are used to fractionate the samples for GC-MS analysis or bioassay. Micropore-free surface-activated carbon is highly selective for PCDD/Fs and non-ortho-PCBs and can improve the conventional activated carbon column clean-up. Along with sulfuric acid-coated diatomaceous earth columns, micropore-free surface-activated carbon provides a rapid, robust, and high-throughput sample preparation method for PCDD/Fs and non-ortho-PCBs analysis.     

Rate constant of the reaction of NO3 with CH2I2 measured with use of cavity ring-down spectroscopy

We have applied cavity ring-down spectroscopy to a kinetic study of the reaction of NO₃ with CH₂I₂ in 25–100 Torr of N₂ diluent at 298 K. The rate constant of reaction of NO₃ + CH₂I₂ is determined to be (4.0 ± 1.2) × 10⁻¹³ cm³ molecule⁻¹ s⁻¹ in 100 Torr of N₂ diluent at 298 K. The rate constant increases with increasing pressure of buffer gas below 100 Torr. The reaction of CH₂I₂ with NO₃ has the potential importance at nighttime in the atmosphere.     

Photografting of vinyl polymers onto ultrafine inorganic particles: Photopolymerization of vinyl monomers initiated by AZO groups introduced onto these surfaces

The photografting of polymers onto ultrafine inorganic particles, such as silica and titanium oxide, initiated by azo groups introduced onto these surfaces was investigated. The introduction of azo groups onto the particles was achieved by the reaction of 4,4′-azobis(4-cyanopentanoic acid) with surface isocyanate groups, which were introduced by the treatment with tolylene 2,4-diisocyanate. It was found that the photopolymerization of vinyl monomers, such as methyl methacrylate (MMA), styrene, and N-vinylcarbazole, is initiated by ultrafine particles having azo groups. The corresponding polymers were effectively grafted onto these surfaces through the propagation of the polymer from the surface radicals formed by the photodecomposition of the azo groups: e.g., the percentage of grafting of PMMA and polystyrene onto silica was reached to 112 and 176%, respectively. The percentage of grafting onto silica in the graft polymerization initiated by photodecomposition of surface azo groups was much larger than that initiated by thermal decomposition. Polymer-grafted ultrafine particles thus obtained gave a stable colloidal dispersion in good solvents for the grafted chain. © 1994 John Wiley & Sons, Inc.