Keisuke Makino

Cautionary note for DMPO spin trapping in the presence of iron ion

2-Hydroxy-5,5-dimethyl-1-pyrrolidinyloxy (DMPO-OH), which is known to be produced by spin trapping of hydroxyl radicals (.OH) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and has been a good monitor for detecting .OH in biological systems, has been examined by EPR for its production scheme in the presence of iron ion. In an aqueous DMPO solution containing ferric ion (Fe3+), DMPO-OH was produced and addition of methanol, a good scavenger for .OH, to this solution led to an aminoxyl radical, DMPO-OCH3, instead of DMPO-CH2OH which is produced by DMPO spin trapping of .CH2OH arising from H-abstraction by .OH. Also EPR measurements at 77K indicated the formation of a chelate between DMPO and Fe3+. Based on these, it has been elucidated that DMPO-OH as well as DMPO-OCH3 is formed by the nucleophilic attack of water and methanol to the chelating DMPO, respectively.

Development of a Novel Artificial Matrix with Cell Adhesion Peptides for Cell Culture and Artificial and Hybrid Organs

The authors present a novel artificial matrix with high cell adhesion and growth rate that was produced by chemical fixation of an RGD-containing peptide (Arg-Gly-Asp) on a polyvinyl alcohol (PVA) surface. The logical sequence behind the molecular design of an artificial matrix that mimics natural adhesive proteins, such as fibronectin, is based on the fact that an RGD tripeptidyl amino sequence is the minimal common adhesion site of adhesive proteins. Activation of surface hydroxyl groups by carbonyl diimidazole successfully incorporated GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro) onto PVA films. The resultant film surface was found to be bioactive, molecularly recognizing the adhesive receptor of bovine endothelial cells. Thus, the artificial matrix mimics active adhesion sites and serves as a novel artificial matrix that may be useful for cell culture, tissue-compatible implants, and hybrid artificial organs.

Essential Oil Phenyl Propanoids. Useful as OH Scavengers

In order to search for radical scavengers which could be used as raw materials for cosmetics, phenyl propanoids (eugenol, isoeugenol, dehydrodieugenol, dehydrodieugenol B and coniferyl aldehyde) were examined for their hydroxyl radical (.OH) scavenging ability. A Fenton system was used to produce .OH. In order to see scavenging by these phenyl propanoids, competition reactions between a spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), and these phenyl propanoids for .OH were studied. The relative yield of the spin adduct of .OH (DMPO-OH) was measured by electron spin resonance spectroscopy. The approximate rate constants of the reactions between these phenyl propanoids and .OH estimated by measuring the reduced height of the ESR signals of DMPO-OH were found to be at least in the order of 10(9) M-1 s-1 (diffusion-controlled). Also, using the TBA tests, the reactions between .OH and several compounds reactive with .OH were investigated in the presence of the phenyl propanoids and it was found that the phenyl propanoids compete with such reactive compounds for .OH. These results indicate that these phenyl propanoids can be used as antioxidants for skin damage perhaps caused by .OH generated by UV-light.

Preparation and chromatographic characteristics of a chiral-recognizing perphenylated cyclodextrin column

Abstract A novel bifunctional chiral recognizing column (PhCD column) was prepared by immobilizing, on silica gel, β-cyclodextrin (CD) which was perphenylated through the reaction of phenyl isocyanate. Using several enantiomeric compounds, chiral recognition by CD was determined to be maintained in a PhCD column. The chromatographic characteristics of a PhCD column were compared with those of a column (CD column) bound with underivatized CD, and the following properties were found. For polyaromatic compounds and alkylbenzene derivatives, hydrophobic interaction is predominant, and for phenylalkyl alcohols and amines whose enatiomers are hard to recognize on a CD column, specific enantioselectivity was exhibited under the effects of the chemical structure around the chiral centre of the solutes. This added function is possibly an induced fit caused by the phenyl cluster on the CD openings. Using these properties, various enantiomeric drugs, in particular β-blockers (amino alcohols), which are hardly separated on conventional chiral separation columns, could be separated.

High-performance gel filtration of water-soluble samples on polyvinyl alcohol columns

Abstract The chromatographic behaviour in aqueous solution of pullulans, polyethylene glycols, peptides and proteins with different molecular weights on newly developed polyvinyl alcohol columns (Asahipak GS column series: GS-310, GS-320, GS-510 and GS-520) has been investigated. Pullulans and polyethylene glycols eluted according to the gel filtration mode, so both series of compounds produced almost equal calibration curves for GS-310 and GS-320 and for GS-510 and GS-520; the exclusion limits on GS-310 and GS-320 and on GS-510 and GS-520 were found to be ca. 40,000 and 300,000, respectively. Peptides and proteins were found to adsorb slightly on the columns. However, the plots of the elution volumes against the logarithm of the molecular weights for many of the substrates tested gave rise to the linear calibration curves. Recovery of several crude proteins was also studied for the columns and high values were obtained (81–100%). The effects of changes in the flow-rate, temperature and concentration of electrolytes added to the eluents, on the retention of all four types of compound are also reported.

Spin-Labeled Oligonucleotides Site Specifically Labeled at the Internucleotide Linkage. Separation of Stereoisomeric Probes and EPR Spectroscopical Detection of Hybrid Formation in Solution

Abstract Described herein is the synthesis, separation of stereoisomers, hybridization, and EPR behavior of spin-labeled oligonucleotides site specifically labeled at the internucleotide linkage with 4-amino-TEMPO. The results obtained indicate that the spin-labeling method will provide a DNA probe useful for detecting hybrid formation with the complementary segment in solution.

Structure of the active 27-residue fragment of human calpastatin

A synthetic 27‐residue peptide corresponding to exon 1B of the endogenous inhibitor calpastatin contains a well‐conserved region and has an ability to inhibit the cysteine endopeptidase calpain specifically. We examined the solution structure of this peptide in DMSO‐d6 by two‐dimensional 1H NMR spectroscopy. Although regular secondary structures such as α‐helix and β‐sheet were not found, the region from Ile18 to Arg23 formed a well‐defined structure with a type I β‐turn. This region coincided well with the highly conserved region of calpastatin. The result strongly suggests that this turn structure is essential for the inhibitory activity of calpastatin.

An Artifact in the ESR Spectrum Obtained by Spin Trapping with Dmpo

In order to overcome a common problem in spin trapping with high concentrations of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) where ESR spectra are obtained which include an unidentified set of lines composed of a triplet of doublets, commercial DMPO was analyzed for its impurities by high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy. It has been determined that this undesirable ESR spectrum is due to an impurity included in the spin trap. This compound has been assigned to the hydroxylamine which is a DMPO-derivative having an epoxy ring located at the 2 and 3 positions.

An ESR study on lipid peroxidation process. Formation of hydrogen atoms and hydroxyl radicals

Lipid peroxidation process was studied by the combination of ESR spectroscopy and spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide. Autoxidation of egg lecithin phosphatidylcholine dispersed in water was found to produce hydrogen atoms and hydroxyl radicals. It was also found that for producing such reactive species, unsaturated fatty acid moieties are needed. The ESR spectrum obtained from this model system was identical with that from body fluid such as serum, indicating that hydrogen atoms and hydroxyl radicals could be generated in living bodies during the course of lipid peroxidation.

Reversed-phase ion-pair chromatography of oligodeoxyribonucleotides

The separation of large oligodeoxyribonucleotides, synthesized chemically and subsequently deblocked, was carried out by reversed-phase ion-pair chromatography (RP-IPC) with a linear gradient of acetonitrile concentration. It was found that tetrabutylammonium phosphate is suitable as an ion-pairing reagent and produces a linear relationship between the base numbers of the samples and their elution volumes. It was also verified that various factors effective in reversed-phase chromatography, such as temperature, end-capping of the columns, differences in the type of C18 alkylating reagents and in the base silica, and the pore size of the base silica have little effect on the resulting separation by RP-IPC.