Photodissociation of heme distal methionine in ferrous cytochrome C revealed by subpicosecond time-resolved resonance Raman spectroscopy

Cytochrome c (cyt c) is an electron-transfer heme protein that also binds nitric oxide (NO). In resting cyt c, two endogenous ligands of the heme iron are histidine-18 (His) and methionine-80 (Met) side chains, and NO binding requires the cleavage of one of the axial bonds. Previous femtosecond transient absorption studies suggested the photolysis of either Fe-His or Fe-Met bonds. We aimed at unequivocally identifying the internal side chain that is photodissociated in ferrous cyt c and at monitoring heme structural dynamics, by means of time-resolved resonance Raman (TR3) spectroscopy with approximately 0.6 ps time resolution. The Fe-His stretching mode at 216 cm-1 has been observed in photoproduct TR3 spectra for the first time for a c-type heme. The same transient mode was observed for a model ferrous cyt c N-fragment (residues 1-56) ligated with two His in the resting state. Our TR3 data reveal that upon ferrous cyt c photoexcitation, (i) distal Met side chain is instantly released, producing a five-coordinated domed heme structure, (ii) proximal His side chain, coupled to the heme, exhibits distortion due to strain exerted by the protein, and (iii) alteration in heme-cysteine coupling takes place along with the relaxation of the protein-induced deformations of the heme macrocycle.     

Spin-adapted wave functions of the serber type and time reversal

A procedure is proposed for generating Serber-type spin eigenfunctions withM _s = 0. The procedure uses the time-reversal invariance of these functions to increase the efficiency and to reduce the storage requirements. Simplifications in calculating the matrix elements of an observable operator which follow from the use of the time-reversal symmetry are briefly discussed.     

Flow injection-chemical vapor generation atomic fluorescence spectrometry hyphenated system for organic mercury determination: A step forward

Monomethylmercury and ethylmercury were determined on line using flow injection-chemical vapor generation atomic fluorescence spectrometry without neither requiring a pre-treatment with chemical oxidants, nor UV/MW additional post column interface, nor organic solvents, nor complexing agents, such as cysteine. Inorganic mercury, monomethylmercury and ethylmercury were detected by atomic fluorescence spectrometry in an Ar/H₂ miniaturized flame after sodium borohydride reduction to Hg⁰, monomethylmercury hydride and ethylmercury hydride, respectively. The effect of mercury complexing agent such as cysteine, ethylendiaminotetracetic acid and HCl with respect to water and Ar/H₂ microflame was investigated.The behavior of inorganic mercury, monomethylmercury and ethylmercury and their cysteine-complexes was also studied by continuous flow-chemical vapor generation atomic fluorescence spectrometry in order to characterize the reduction reaction with tetrahydroborate. When complexed with cysteine, inorganic mercury, monomethylmercury and ethylmercury cannot be separately quantified varying tetrahydroborate concentration due to a lack of selectivity, and their speciation requires a pre-separation stage (e.g. a chromatographic separation). If not complexed with cysteine, monomethylmercury and ethylmercury cannot be separated, as well, but their sum can be quantified separately with respect to inorganic mercury choosing a suitable concentration of tetrahydroborate (e.g. 10^? 5 mol L^? 1), thus allowing the organic/inorganic mercury speciation.The detection limits of the flow injection-chemical vapor generation atomic fluorescence spectrometry method were about 45 nmol L^? 1 (as mercury) for all the species considered, a relative standard deviation ranging between 1.8 and 2.9% and a linear dynamic range between 0.1 and 5 μmol L^? 1 were obtained. Recoveries of monomethylmercury and ethylmercury with respect to inorganic mercury were never less than 91%. Flow injection-chemical vapor generation atomic fluorescence spectrometry method was validated by analyzing the TORT-1 certificate reference material, which contains only monomethylmercury, and obtaining 83 ± 5% of monomethylmercury recovered, respectively. This method was also applied to the determination of monomethylmercury in saliva samples.     

Development and Application of Ultra Performance Liquid Chromatography Method to the Quantification of the Biotransformation of Methyl Paraben in Eisenia foetida

A simple and quick ultra performance liquid chromatography method (UPLC) has been developed for determination of methyl paraben (MP) and its major metabolites p‐hydroxybenzoic acid (pHBA) and phenol (Phe), following its biotransformations in Eisenia foetida. After different exposure time to paper contact test, the presence of methyl paraben and his biotransformation products in adult earthworms was monitored. Determination of its metabolites was achieved with a BEH (bridged ethane‐silicon hybrid) C₁₈ column (2.1×50 mm i.d., 1.7 µm particle size), using methanol/water/phosphoric acid as mobile phase, under a gradient elution program, and a PDA (photo‐diode array) detection (quantification with MaxPlot in the range 210–400 nm). The absorption of MP did not exceed 30% and in the first 4–6 h after exposure only minute amounts of pHBA and Phe were detected in the worm homogenates. After 48 h of exposures, almost 70% of absorbed MP was already metabolized to Phe and around 20% could be found as pHBA.     

Synthesis of (S)‐3‐heteroaryl‐2‐hydroxy‐l‐propyl benzoates by ‘ring switching’ methodology

(S)‐5‐Benzoyloxymethyl‐3‐[(E)‐(dimethylamino)methylidene]tetrahydrofuran‐2‐one (6), prepared in 5 steps from L‐glutamic acid (1), was used as precursor in a one step ‘ring switching’ synthesis of (S)‐2‐hydroxy‐3‐heteroaryl‐l‐propyl benzoates 13‐18, 23, 24. In the reaction of 6 with 2‐aminopyridine (21) and 2‐amino‐4,6‐dimethylpyrimidine (22) the corresponding dimethylamine substitution products (25, 26) were obtained.     

Site‐Specific Investigation of the Steady‐State Kinetics and Dynamics of the Multistep Binding of Bile Acid Molecules to a Lipid Carrier Protein

The investigation of multi‐site ligand–protein binding and multi‐step mechanisms is highly demanding. In this work, advanced NMR methodologies such as 2D ¹H–¹⁵N line‐shape analysis, which allows a reliable investigation of ligand binding occurring on micro‐ to millisecond timescales, have been extended to model a two‐step binding mechanism. The molecular recognition and complex uptake mechanism of two bile salt molecules by lipid carriers is an interesting example that shows that protein dynamics has the potential to modulate the macromolecule–ligand encounter. Kinetic analysis supports a conformational selection model as the initial recognition process in which the dynamics observed in the apo form is essential for ligand uptake, leading to conformations with improved access to the binding cavity. Subsequent multi‐step events could be modelled, for several residues, with a two‐step binding mechanism. The protein in the ligand‐bound state still exhibits a conformational rearrangement that occurs on a very slow timescale, as observed for other proteins of the family. A global mechanism suggesting how bile acids access the macromolecular cavity is thus proposed.     

Annealing study of two-dimensional patterned Ge nanostructures via nanosphere lithography

Experimental studies on patterning hexagonal Ge nanostructures have been conducted on Si substrates through deposition of Ge with colloidal particles as a mask. The deposited Ge thin film possesses, according to the X-ray diffraction measurements, in plane texture, being epitaxial and aligned with the (111) Si substrate. The size distribution of the patterned Ge nanostructures is narrow, as indicated by the atomic force microscopy and scanning electron microscopy measurements. We have obtained Ge nanostructures with lateral dimension of 490 nm (height 12 nm), 200 nm (height 6 nm) and 82 nm (height 6 nm) by using different sizes of polystyrene spheres. We have performed in depth studies of the Ge nanostructures’ behavior due to thermal and rapid thermal post-annealing processes. FT micro-Raman spectroscopy shows that there is no Si intermixing during the annealing process. In order to quantify the changes in the height and lateral dimension, we have performed atomic force microscopy and white light interferometry analysis. The changes in shape and the decrease in the area of a cross-section of Ge nanostructure will be discussed in respect to similar results shown in the literature for Ge thin films during the annealing process.     

The stochastic bid generation problem in combinatorial transportation auctions

In this paper, we deal with the generation of bundles of loads to be submitted by carriers participating in combinatorial auctions in the context of long-haul full truckload transportation services. We develop a probabilistic optimization model that integrates the bid generation and pricing problems together with the routing of the carrier’s fleet. We propose two heuristic procedures that enable us to solve models with up to 400 auctioned loads.     

Variational technique and principal solution in half-linear oscillation criteria

We investigate oscillatory properties of half-linear differential equations. The investigated half-linear equation is viewed as a perturbation of another (nonoscillatory) equation of the same form and perturbation is allowed both in the absolute term and in the derivative term. First, the Sturmian type theorem for solutions of the associated Riccati equation is established. In the second part of the paper we prove new half-linear oscillation criteria using the variational technique.