Rapid purification of detergent-solubilized bovine hormone-sensitive lipase by high performance hydrophobic interaction chromatography

Hormone-sensitive lipase (HSL), the enzyme controlling the rate of adipose tissue lipolysis and also possibly involved in the regulation of steroidogenesis, has been purified from bovine omental adipose tissue. Partially detergent-solubilized, delipidated and purified HSL was obtained through step-elution at conventional DEAE ion-exchange chromatography, followed by concentration on hydroxylapatite. High performance hydrophobic interaction chromatography (HPHIC) on phenylsilica then resulted in an increase of HSL protein purity from 2% to more than 70%. Final purification of the enzyme to apparent homogeneity (greater than 95% protein purity), concentration and removal of most of the detergent was obtained by high performance cation exchange chromatography on Mono S. At least 0.5 mg of highly stable HSL was obtained from 5 kg of bovine omental fat within four working days. The purified lipase had a lower specific activity than previously reported for the corresponding rat enzyme but the preparations have proved very useful for enzyme structure studies and as an antigen.     

Decomposition of acoustic fields in quantised Bessel beams

Bessel beams are non-diffracting solutions to the wave equation, which become limited diffraction beams when implemented on finite apertures. In previous work we have applied Fourier-Bessel theory to deduce theoretically that the quantization of the Bessel beam profile on annular arrays results in a field which is a sum of limited diffraction fields. Here we demonstrate this for a five-ring, 20-mm diameter, 2.5-MHz transducer. The quantized field comprises a weighted sum of a main component corresponding to the desired field, along with three other major components and 28 lesser components representing undesired field components. The three major components correspond to limited diffraction beams with narrower beamwidths and shorter depths of field than the desired beam, and we show that these account for most of the discrepancies between the desired field and actual quantized field. An estimate and an interpretation of the number of field components as a function of the wavenumber are also given.     

Recovery of reflectance spectra from colorimetric data using principal component analysis embedded regression technique

The classical principal component analysis technique is enhanced for reconstruction of reflectance spectra of surface colors from the corresponding tristimulus values under a given set of viewing conditions, i.e., D65 illuminant and 1964 standard observer. In this paper, the number of implemented eigenvectors has been virtually extended from three to six by estimation of another set of tristimulus values under illuminant A and 1964 standard observer. The second set of colorimetric data was predicted by the conventional non-linear regression method and used in the spectral reconstruction to produce a fully determined system in the case of six eigenvectors. The improvement obtained from the proposed modification was examined for the recovery of the reflectance spectra of Munsell color chips as well as ColorChecker DC samples. The performance is evaluated by the mean, maximum and standard deviation of color difference values under other sets of light sources. The values of mean, maximum and standard deviation of root mean square (RMS) errors between the reproduced and the actual spectra were also calculated. Results are compared with those obtained from traditional methods using the principal component analysis (PCA) routine. All metrics show that the suggested method leads to considerable improvements in comparison with the standard PCA approach.     

Sulfur ligand substitution at the nickel(II) sites of cubane-type and cubanoid NiFe3S4 clusters relevant to the C-clusters of carbon monoxide dehydrogenase

Substitution reactions at the nickel site of the cubane-type cluster [(Ph3P)NiFe3S4(LS3)]2- (2) have been investigated in the course of a synthetic approach to the C-clusters of CODH. Reaction of 2 with RS- or toluene-3,4-dithiolate affords [(RS)NiFe3S4(LS3)]3- (R = Et (5), H (6)) or [(tdt)NiFe3S4(LS3)]3- (7), demonstrating that anionic sulfur ligands can be bound at the NiII site. Clusters 5 and 6 contain tetrahedral Ni(micro3-S)3(SR) sites. Cluster 7 is of particular interest because it includes a cubanoid NiFe3(micro2-S)(micro3-S)3 core and an approximately planar Ni(tdt)(micro3-S)2 unit. The cubanoid structure is found in all C-clusters, and an NiS4-type unit has been reported in C. hydrogenoformans CODH. Clusters 5/6 are formulated to contain the core [NiFe3S4]1+ identical with Ni2+ (S = 1) + [Fe3S4]1- (S = 5/2) and 7 the core [NiFe3S4]2+ identical with Ni2+ (S = 0) + [Fe3S4]0 (S = 2) on the basis of structure, 57Fe isomer shifts, and 1H NMR isotropic shifts. Also reported are [(EtS)CuFe3S4(LS3)]3- (9) and [Fe4S4(LS3)(tdt)]3- (11). The structures of 5-7, 9, and 11 are presented. Cluster 11, with a five-coordinate Fe(tdt)(micro3-S)3 site, provides a clear structural contrast with 7, which is currently the closest approach to a C-cluster but lacks the exo iron atom found in the NiFe4S4,5 cores of the native clusters. (CODH = carbon monoxide dehydrogenase, LS3 = 1,3,5-tris((4,6-dimethyl-3-mercaptophenyl)thio)-2,4,6-tris(p-tolylthio)benzene(3-), tdt = toluene-3,4-dithiolate).     

On-line SPE-Nano-LC-Nanospray-MS for rapid and sensitive determination of perfluorooctanoic acid and perfluorooctane sulfonate in river water

An instrumental set up including on-line solid-phase extraction, nano-liquid chromatography, and nanospray mass spectrometry is constructed to improve the sensitivity for quantitation of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) in surface water. Sample volumes of 1000 microL are loaded onto a microbore 1.0-mm i.d. x 5 mm, 5 microm Kromasil C(18) enrichment column by a carrier solution consisting of 10mM ammonium acetate in acetonitrile-water (10:90, v/v) at a flow rate of 250 microL/min, providing on-line analyte enrichment and sample clean-up. Backflushed elution onto a 0.1-mm i.d. x 150 mm, 3.5 microm Kromasil C(18) analytical column is conducted using an acetonitrile-10mM ammonium acetate solvent gradient from 30% to 70% acetonitrile. Water samples are added with internal standard (perfluoroheptanoic acid) and filtrated prior to injection. The mass limits of detection of PFOA and PFOS are 0.5 and 1 pg, respectively, corresponding to concentration limits of detection of 500 pg/L and 1 ng/L, respectively. The total time spent on sample preparation, chromatography, and detection is approximately 12 min per sample. The method was employed for the determination of PFOS and PFOA in urban river water.     

Decomposition of ruthenium olefin metathesis catalysts

The decomposition of a series of ruthenium metathesis catalysts has been examined using methylidene species as model complexes. All of the phosphine-containing methylidene complexes decomposed to generate methylphosphonium salts, and their decomposition routes followed first-order kinetics. The formation of these salts in high conversion, coupled with the observed kinetic behavior for this reaction, suggests that the major decomposition pathway involves nucleophilic attack of a dissociated phosphine on the methylidene carbon. This mechanism also is consistent with decomposition observed in the presence of ethylene as a model olefin substrate. The decomposition of phosphine-free catalyst (H2IMes)(Cl)2Ru=CH(2-C6H4-O-i-Pr) (H2IMes = 1,3-dimesityl-4,5-dihydroimidazol-2-ylidene) with ethylene was found to generate unidentified ruthenium hydride species. The novel ruthenium complex (H2IMes)(pyridine)3(Cl)2Ru, which was generated during the synthetic attempts to prepare the highly unstable pyridine-based methylidene complex (H2IMes)(pyridine)2(Cl)2Ru=CH2, is also reported.     

Generalized frieze pattern determinants and higher angulations of polygons

Frieze patterns (in the sense of Conway and Coxeter) are in close connection to triangulations of polygons. Broline, Crowe and Isaacs have assigned a symmetric matrix to each polygon triangulation and computed the determinant. In this paper we consider d-angulations of polygons and generalize the combinatorial algorithm for computing the entries in the associated symmetric matrices; we compute their determinants and the Smith normal forms. It turns out that both are independent of the particular d  -angulation, the determinant is a power of d−1$d-1$, and the elementary divisors only take values d−1$d-1$ and 1. We also show that in the generalized frieze patterns obtained in our setting every adjacent 2×2$2\times 2$-determinant is 0 or 1, and we give a combinatorial criterion for when they are 1, which in the case d=3$d=3$ gives back the Conway–Coxeter condition on frieze patterns.