Salvatore Fisichella

Dimerization of Cibacron Blue F3GA and other dyes: influence of salts and temperature

Abstract The monomer–dimer equilibria of Cibacron Blue F3GA (CB) and five other dyes (Levafix Brilliant Blue EB, Reactive Scarlet 017, Methyl Orange, Basic Blue 3 and Chicago Blue Sky) have been investigated in water and in the presence of KH 2 PO 4 . Aggregation of CB has been also examined in the presence of NaH 2 PO 4 , LiCl and KCl. When a new iterative approach, based on non-linear least-square (NLLSQ) fitting procedure was applied, it was found that the dimerization constants depend on the extension of organic molecules and the number of sulphonic groups. In the case of CB, cations had a greater effect on the equilibrium than anions. Analysis of the calculated spectra for monomer and dimer of Basic Blue 3 after deconvolution allowed us to specify the geometry of the dimer.

Protective Effects of L- and D-Carnosine on α-Crystallin Amyloid Fibril Formation: Implications for Cataract Disease

Mildly denaturing conditions induce bovine alpha-crystallin, the major structural lens protein, to self-assemble into fibrillar structures in vitro. The natural dipeptide l-carnosine has been shown to have potential protective and therapeutic significance in many diseases. Carnosine derivatives have been proposed as potent agents for ophthalmic therapies of senile cataracts and diabetic ocular complications. Here we report the inhibitory effect induced by the peptide (l- and d-enantiomeric form) on alpha-crystallin fibrillation and the almost complete restoration of the chaperone activity lost after denaturant and/or heat stress. Scanning force microscopy (SFM), thioflavin T, and a turbidimetry assay have been used to determine the morphology of alpha-crystallin aggregates in the presence and absence of carnosine. DSC and a near-UV CD assay evidenced that the structural precursors of amyloid fibrils are polypeptide chain segments that lack stable structural elements. Moreover, we have found a disassembling effect of carnosine on alpha-crystallin amyloid fibrils. Finally, we show the ability of carnosine to restore most of the lens transparency in organ-cultured rat lenses exposed to similar denaturing conditions that were used for the in vitro experiments.

A new highly diastereoselective synthesis of epi-inositol from d-galactose

Abstract The inosose derivative 3 was obtained with high stereoselectivity by intramolecular aldol condensation of the aldohexos-5-ulose derivative 2 , and it was selectively reduced and debenzylated to give epi -inositol in high yield. The stereochemistry and the preferred conformations of compounds 3 – 7 were determined through 1D and 2D NMR experiments.

Conformational analysis of some (E)-α-phenyl-β-(2-thienyl)- and -(2-furyl_acrylic acids

Abstract NMR spectral data of some (E)-α-phenyl-β-(2-thienyl) acrylic acids indicate that these compounds exist in the preferred s-trans conformation. In the case of (E)-α-phenyl-β-(2-furyl)acrylic acids and their methyl esters the presence of only s-cis rotamer has been established.

Isolation by gel-permeation chromatography of a non-covalent complex of Cibacron Blue F3G-A with human serum albumin.

The isolation by gel-permeation chromatography on Sephadex G-100 of a non-covalent complex of Cibacron Blue F3G-A (CB) with human serum albumin (HSA) is described. The complex presents a molar ratio of 3:1 CB-HSA and can be re-chromatographed under the same conditions without modification of its composition. However, complete dissociation occurs when the complex is chromatographed in the presence of denaturing agents. The effect of pH on the molar composition of the complex was also investigated by gel-permeation chromatography. Analogous complexes between CB and A and C cyanogen bromide fragments of unreduced HSA were also isolated by gel-permeation chromatography on Sephadex G-50. They present a molar ratio of 0.8:1 and 1.3:1 CB-protein for fragments A and C, respectively. These results suggest that two of the three molecules of CB bound to HSA may be located in the hydrophobic pocket corresponding to subdomain IIA, with the other molecule in the hydrophobic site corresponding to subdomain IIIA. The UV-Vis and dichroic circular spectra of the isolated complexes are reported.

Purification of wheat flour high-Mr glutenin subunits by Reactive Red 120-Agarose and Reactive Yellow 86-Agarose resins

Abstract Two reactive textile dyes were employed to purify some high-Mr glutenin subunit mixtures by dye-ligand chromatography. In particular, the use of the triazine dye Reactive Red 120 coupled to Agarose bed provided an efficient method to purify high-Mr glutenin subunit mixtures, showing high binding capacity and high selectivity for these proteins and assuring a recovery of about 80%. On the other hand, the Reactive Yellow 86 had weaker interaction and a lower selectivity than Red 120 with a recovery of about 60%. However, this dye allowed the separation of some subunits that were not separated by reactive Red 120.

Use of hydroxyacetophenones as matrices for the analysis of high molecular weight glutenin mixtures by matrix-assisted laser desorption/ionization mass spectrometry

A selection of hydroxyacetophenones has been investigated as matrices for the analysis, by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), of high molecular weight (HMW) glutenin mixtures from three wheat varieties. Mass spectra were obtained directly from the HMW glutenin extracts without any preliminary purification and separation steps. According to the quality of the mass spectra, obtained using different hydroxyacetophenones, it has been possible to classify the matrices on the basis of their suitability for the analyte properties. Only two of the matrices considered showed to be compatible with the HMW glutenin mixtures analysis, although a large amount of other highly complex protein mixtures are present. This study indicates that MALDI-MS, as a stand-alone technique, is particularly useful for the direct determination of the complete HMW subunits profile and their molecular weights. Copyright 1999 John Wiley & Sons, Ltd.

Chromatographic profiles of cyanogen bromide fragments of unreduced human serum albumin on immobilized Cibacron Blue F3G-A

Abstract The elution profiles of cyanogen bromide fragments A (299–585), B (1–123), C (124–298) and D (1–298) of unreduced human serum albumin (HSA) on Cibacron Blue F3G-A immobilized on Sepharose CL-6B are reported. The binding properties of fragments C and D are similar to those of HSA, whereas fragment A shows a slightly lower retention time. Fragment B, in contrast, does not interact with the dye. The different chromatographic behaviour of fragments B and C allows their fast separation by combined use of gel permeation and dye-protein affinity chromatography.

Adsorption isotherms on cotton of direct sky blue FF from aqueous ethanolic solutions

Abstract Adsorption isotherms at 80° Cfor Direct Sky Blue FF (C.I. Direct Blue 1) on cotton in the presence of variable amounts of NaCl (2, 10, 15 and 50 g litre−1) and ethanol (v/v) (0, 5, 10, 20, 30, 40, 50, 60%) were measured. The Langmuir equation has been used to describe the equilibrium except for the measurements obtained at 15 g litre−1 of NaCl and 50 % and 60 % of ethanol, and at 50 g litre−1 of NaCl and 20 % of ethanol for which the distribution law was followed. The saturation values increased markedly with the salt concentration. The concentration of ethanol influenced the adsorption in two ways; one was a depressing action and the other an accelerating action. The increasing concentration of ethanol decreased the degree of association of the dye and the adsorption was Langmuir type; at 15glitre−1 ofNaCl and 50% and 60% ofethanol,and 50 g litre −1 of NaCl and 20% of ethanol the degree of association of the dye increased and the adsorption was distribution type.

Tryptic peptide mapping of sequence 299-585 of human serum albumin by high-performance liquid chromatography and fast atom bombardment mass spectrometry

The determination of the tryptic peptide mapping of sequence 299-585 (cyanogen bromide fragment A) of human serum albumin (HSA) by chemical and enzymatic cleavages and combined use of HPLC and FAB-MS is described. Reduction and carboxymethylation of A gave four subfragments which were separated by HPLC and digested with trypsin. Tryptic fragments were separated by HPLC and identified by FAB-MS. A total coverage of about 95% of the entire sequence was obtained. Tryptic fragments not identified include mostly single amino acids and very hydrophilic peptides which were absent in the chromatograms. The high reproducibility of the experiments and the satisfactory yield of the tryptic fragments identified demonstrate the great potential of the combined use of HPLC separation and FAB-MS analysis for the structural investigation of HSA.