Protein secondary structure and stability determined by combining exoproteolysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

In the post-genomic era, several projects focused on the massive experimental resolution of the three-dimensional structures of all the proteins of different organisms have been initiated. Simultaneously, significant progress has been made in the ab initio prediction of protein three-dimensional structure. One of the keys to the success of such a prediction is the use of local information (i.e. secondary structure). Here we describe a new limited proteolysis methodology, based on the use of unspecific exoproteases coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), to map quickly secondary structure elements of a protein from both ends, the N- and C-termini. We show that the proteolytic patterns (mass spectra series) obtained can be interpreted in the light of the conformation and local stability of the analyzed proteins, a direct correlation being observed between the predicted and the experimentally derived protein secondary structure. Further, this methodology can be easily applied to check rapidly the folding state of a protein and characterize mutational effects on protein conformation and stability. Moreover, given global stability information, this methodology allows one to locate the protein regions of increased or decreased conformational stability. All of this can be done with a small fraction of the amount of protein required by most of the other methods for conformational analysis. Thus limited exoproteolysis, together with MALDI-TOF MS, can be a useful tool to achieve quickly the elucidation of protein structure and stability.     

High stability amperometric biosensor based on enzyme entrapment in microgels

The preparation and characterization of an amperometric glucose biosensor based on the entrapment of glucose oxidase (GOx) in a polyacrylamide microgel is described. This study proves that polyacrylamide microgels provide an excellent matrix for GOx immobilization that can be used as a biological material in amperometric biosensors. The interference produced by ascorbic and uric acid has been eliminated by including acrylic acid in the polymeric matrix. With this modification, we obtain an adequate device for glucose determination in complex samples such as blood and serum. The study of the temperature effect in the response of biosensors indicates that swelling of the microgels directly influences the enzymatic activity. Thus, the behaviour of the enzyme in the swollen microgels is similar to the enzyme in solution, but the enzyme's activation energy increases when the water content in the microgels decreases. One important property of these biosensors is their remarkable stability. After 4 months of its manufacture, there is no loss in the initial response. Furthermore, the enzymatic activity of freeze-dried microgels containing enzyme remains unaltered for at least 18 months.     

Graft polymerization of acrylic monomers onto starch fractions. I. Effect of reaction time on grafting methyl methacrylate onto amylose

A study was made of the ceric-ammonium-nitrate-initiated graft polymerization of methyl methacrylate (MMA) onto a lineal fraction of starch (amylose). Grafting yields were determined by extraction with appropriate solvents: 1,2-dichloroethane for homo-PMM, and 0.5N NaOH for ungrafted amylose. Percent grafting was calculated by acid hydrolysis with 1N HCl. Molecular weights of PMMA side chains were determined by gel permeation-chromatography. Grafting efficiencies ranged from 72 to 83%, grafting, from 190 to 271%, and frequency of attachment of side chains, from 900–1250 glucose units per chain. The results observed are discussed.     

Conformational behaviour of 2,2′-bipyrrole

The rotational potential around the interannular bond in 2,2-bipyrrole has been calculated making use of standard minimal STO-3G and split valence 4-31G basis sets. Geometrical optimization concerning the most significant interannular internal parameters has been performed with both basis sets. The trans conformer is predicted to be more stable than the cis. The minimal basis set predicts the existence of a cisoid-gauche minimum which after limited optimization becomes very shallow and it seems to be an artifact of the rigid rotor approximation. At 4-31G level, both the trans and cis conformers represent maxima in the potential curve and two gauche minima appear at =46.0° and =147.6°, the latter being the absolute minimum. The absolute maximum of the potential curve corresponds to the cis conformer.     

Catalysis by supported gold: correlation between catalytic activity for CO oxidation and oxidation states of gold

X-ray absorption near-edge spectra and temperature-programmed oxidation and reduction data demonstrate that Au(I) and Au(0) are both present in working MgO-supported gold catalysts for CO oxidation. EXAFS data indicate gold clusters with essentially the same average diameter (about 30 A) in each catalyst sample. Thus, the results provide no evidence of an effect of gold cluster size on the catalytic activity, but both the catalytic activity and the surface concentration of Au(I) were found to decrease with increasing CO partial pressure (as Au(0) was increasingly formed), demonstrating that the catalytic sites incorporate Au(I).     

Experimental and DFT study of the aza-Diels-Alder reaction between cyclopentadiene and protonated benzylimine derivated from glyoxylates

The Diels-Alder reaction of protonated N-benzyl imine of methyl glyoxylate with cyclopentadiene in different solvents gave mixtures of exo/endo adducts. The exo/endo selectivity of the reaction was elucidated by NMR experiments. Theoretical calculations by means of density functional theory (DFT) at the B3LYP/6-31G(d) level have also been performed to elucidate the molecular mechanism of this reaction. The DFT results suggest a highly asynchronous concerted mechanism, which in turn can explain the preferred exo stereoselectivity of the reaction. Inclusion of solvent effects enhances the exo selectivity, and this effect increases with the polarity of the solvent, in good agreement with the experimental findings.     

Coordination de la phosphine organo-fer [CpFeIIC6Me5CH2PPh2]+ au RhI et proprietes spectroscopiques,electrochimiques et catalytiques des complexes heterobinucleaires FeII-RhI

The reactions between the phosphine-organoiron [CpFe^II-η⁶-C₆Me₅CH₂PPh₂]⁺ PF₆^? (1) and [RhCl(η⁴-diolefin)(μ-Cl)]₂ in CH₂Cl₂ at reflux give the new heterobinuclear air-stable crystalline complexes [CpFe^II-η⁶-C₆Me₅CH₂)P(Ph)₂Rh(η⁴-diene)Cl]PF₆,(D'*-diene=cyclooctadiene (COD): 65%, 2; trimethylfluorobenzobicyclo[2.2.2]octadiene (Me₃TFB): 48%, 3). Complexes 2 and 3 have been studied by ¹H, ¹³C and ³¹P NMR spectroscopy and they are carbonylated (CO, 1 atm). Cyclic voltammetry experiments with addition of MeOH show electron transfer Fe^IRh^I → Fe^IIRh⁰, the presence of a catalytic wave Fe^I/Fe^II and the possible formation of Rh hydrides. Under normal conditions 2 is a catalyst for hydrogenation of cyclohexene, but it is less efficient than the known mononuclear Rh¹ analogues.     

Isotropization in Bianchi type IX vacuum cosmology

We find the most general Bianchi type IX solution in Brans-Dicke theory (BDT) for the vacuum case, with the local rotational symmetry. For BDT coupling parameterw > 500 the universe becomes isotropic for any amount of initial anisotropy. In the extended inflation scenario, the Brans-Dicke scalar field can avoid the inflation in one direction.     

Effect of particle geometry on phase transitions in two-dimensional liquid crystals

Using a version of density-functional theory which combines Onsager approximation and fundamental-measure theory for spatially nonuniform phases, we have studied the phase diagram of freely rotating hard rectangles and hard discorectangles. We find profound differences in the phase behavior of these models, which can be attributed to their different packing properties. Interestingly, bimodal orientational distribution functions are found in the nematic phase of hard rectangles, which cause a certain degree of biaxial order, albeit metastable with respect to spatially ordered phases. This feature is absent in discorectangles, which always show unimodal behavior. This result may be relevant in the light of recent experimental results which have confirmed the existence of biaxial phases. We expect that some perturbation of the particle shapes (either a certain degree of polydispersity or even bimodal dispersity in the aspect ratios) may actually destabilize spatially ordered phases thereby stabilizing the biaxial phase.     

Reactions of thiosemicarbazones derived from beta-keto amides and beta-keto esters with Zn(II) and Cd(II) acetates: influence of metal, substitution, reagent ratio and temperature on metal-induced cyclization

Zinc(II) and cadmium(II) acetates were reacted in methanol under various experimental conditions with thiosemicarbazones derived from beta-keto amides or beta-keto esters (HTSC). Some of these reactions afforded thiosemicarbazonate complexes [M(TSC)2] with IR and NMR spectra compatible with N,S-coordination, but most gave complexes [ML2], where HL is a substituted 2,5-dihydro-5-oxo-1H-pyrazole-1-carbothioamide resulting from cyclization of the HTSC. Some of these pyrazolonates and two of the HL ligands were studied by X-ray diffractometry, and their structures are discussed. Surprisingly, the reactions of zinc(II) acetate with HTSC in 1:1 mol ratio usually gave a third, previously unreported type of complex with a dideprotonated ligand, [Zn(L-H)], which was also formed when [ZnL2] and Zn(OAc)2 interacted at room temperature in 1:1 mol ratio. These L-H complexes are highly insoluble in all common solvents, which hinders their characterization but suggests that they are polymeric in nature.