Spin dynamics in RENi5 ferromagnets byμSR measurements

We report a zero fieldSR study of single crystals of theRENi ₅ ferromagnets where RE=Gd, Er, Dy or Tm. Our work points out the strong influence of the crystal electric field on the rare earth total momentum dynamics: whereas the spin-lattice relaxation process belowT _c is controlled by a two magnon mechanism for a weakly anisotropic magnet (GdNi ₅), the phonons drive the relaxation for a strongly anisotropic magnet such asErNi ₅. ForTmNi ₅ a comparison is made between the fluctuation time measured bySR and¹⁶⁹ Tm Mössbauer spectroscopy.     

The sensitising effect of a sunscreening agent, p-aminobenzoic acid, on near UV induced damage in a repair deficient strain of Escherichia coli.

Abstract. The use of repair deficient strains of bacteria for detecting mutagenic properties of chemical species is now an established technique. In this paper we present inactivation results obtained with Escherichia coli K12 AB2480 (uvr A rec A) which indicate that p-aminobenzoic acid, a common component of sunscreening formulations, may increase the frequency of lethal damage induced in DNA when cell populations are exposed to near ultraviolet radiation. Preliminary experiments, utilising the selective action of photoenzymatic repair, indicated that the sensitisation to near ultraviolet radiation is partially but not wholly due to increased formation of pyrimidine dimers.     

Ion-exchange high-performance liquid chromatographic purification of bovine cardiac and rabbit skeletal muscle troponin subunits

Bovine cardiac and rabbit skeletal troponin complexes were separated into their respective subunits employing high-performance liquid chromatographic (HPLC) techniques on CM-300 and Q-300 ion-exchangers. Bovine cardiac and rabbit skeletal subunits were separated on the strong anion-exchanger, Q-300, in 8 M urea, 50 mM Tris, 2 mM EGTA, 0.5 mM dithiothreitol, pH 7.5, employing a linear salt gradient and on the weak cation-exchanger, CM-300, in 8 M urea, 50 mM potassium dihydrogen phosphate, 2 mM EGTA, 0.5 mM dithiothreitol, pH 6.5, using a linear salt gradient. To obtain complete purification of all components of troponin both ion-exchangers were required. The initial separation of troponin was carried out on the strong anion-exchanger followed by weak cation-exchange chromatography of the troponin I collected from the strong anion-exchange column. The troponin T subunits obtained from Q-300 chromatography demonstrated heterogeneity (three components: T1, T2 and T3) while the troponin I collected from both sources on the Q-300 column were both resolved into major doublets (I1 and I2) when rechromatographed on the CM-300 column. The three troponin T fractions and two troponin I fractions isolated from ion-exchange HPLC were examined by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis to confirm that the heterogeneity was due to differences in charge and not molecular weight. These results were in agreement with the charge differences observed from retention times on ion-exchange HPLC. When comparing the same troponin subunit from different muscle sources, considerable differences in the content of charged amino acid residues were also observed.     

Capillary electrophoresis of amphipathic alpha-helical peptide diastereomers

We have made a rigorous assessment of the ability of capillary electrophoresis to resolve peptide diastereomers through its application to the separation of a series of synthetic 18-residue, amphipathic alpha-helical monomeric peptide analogues, where a single site in the centre of the hydrophobic face of the alpha-helix is substituted by 19 L- or D-amino acids. Such L- and D-peptide pairs have the same mass-to-charge ratio, amino acid sequence and intrinsic hydrophobicity, varying only in the stereochemistry of one residue. CE approaches assessed in their ability to separate diastereomeric peptide pairs included capillary zone electrophoresis (uncoated capillary), micellar electrokinetic chromatography (uncoated capillary in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, CHAPS), open-tubular capillary electrochromatography (C(8)-coated capillary in the presence of 25% 2,2,2-trifluoroethanol (TFE) or 25% ethanol). Overall, the OT-CEC methods were the most effective at separating the most peptide pairs, particularly for those containing hydrophilic side chains. However, the MEKC approach proved most effective for separation of peptide pairs containing hydrophobic or aromatic side chains.     

Preparative reversed-phase liquid chromatography of peptides. Isocratic two-step elution system for high loads on analytical columns

We have developed further our novel sample displacement chromatography (SDC) methodology to carry out preparative separations on analytical equipment and 15-cm analytical columns for sample loads < or = 200 mg. Thus, a two-step isocratic SDC protocol was developed and applied to the purification of important biologically active peptides, i.e. bradykinin antagonists of 10 and 11 residues. Following sample loading in 100% aqueous solvent at a concentration of approximately 7-10 mg/ml (with sample loads varying from 67 to 200 mg) onto a small C18 column (150 x 4.6 mm I.D., made up of three 50-mm columns attached in series), we applied isocratic elution with aqueous acetonitrile at two concentrations, the first (lower concentration) to displace hydrophilic impurities off the column and the second (higher concentration) to displace pure product from the column; hydrophobic impurities remain trapped on the column. This modified SDC approach promises to allow great flexibility in purifying peptides, at high yield of pure product (> 99% purity), and encompassing a range of sample hydrophobicities as well as sample loads (< or = 200 mg) varying by as much as a factor of three.     

Selectivity differences in the separation of amphipathic alpha-helical peptides during reversed-phase liquid chromatography at pHs 2.0 and 7.0: effects of different packings, mobile phase conditions and temperature

In an ongoing effort to understand the effect of varying reversed-phase high-performance liquid chromatography (RP-HPLC) parameters on the retention behaviour of peptides, necessary for the rational development of separation/optimization protocols, we believe it is important to delineate the contribution of alpha-helical structure to the selectivity of peptide separations. The present study reports the effects of varying column packing, mobile phase conditions and temperature on RP-HPLC retention behaviour at pHs 2.0 and 7.0 of peptides based on the amphipathic peptide sequence Ac-EAEKAAKEXEKAAKEAEK-amide (with position X in the centre of the hydrophobic face of the alpha-helix), where position X is substituted by L- or D-amino acids. At pH 2.0, an increase in trifluoroacetic acid concentration or the addition of sodium perchlorate to a phosphoric acid-based mobile phase had the similar effect of improving peak shape as well as increasing peptide retention time due to ion-pairing effects with the positively-charged peptides; in contrast, at pH 7.0, the addition of salt had little effect save an improvement in peak shape. Temperature was shown to have a complex influence on peptide selectivity due to varying effects on peptide conformation. In addition, subtle effects on peptide selectivity were also noted based on the column packings employed at pHs 2.0 and 7.0.