Inhibitor binding to metal-substituted metalloenzyme: Sulfonamide affinity for carbonic anhydrase IX.

Transition metal ions are structural and catalytic cofactors of many proteins including human carbonic anhydrase (CA), a Zn-dependent hydrolase. Sulfonamide inhibitors of CA recognize and form a coordination bond with the Zn ion located in the active site of the enzyme. The Zn ion may be removed or substituted with other metal ions. Such CA protein retains the structure and could serve as a tool to study metal ion role in the recognition and binding affinity of inhibitor molecules. We measured the affinities of selected divalent transition metal ions, including Mn, Fe, Co, Ni, Cu, Cd, Hg, and Zn to metal-free CA isozymes CA I, CA II, and CAIX by fluorescence-based thermal shift assay, prepared metal-substituted CAs, and determined binding of diverse sulfonamide compounds. Sulfonamide inhibitor binding to metal substituted CA followed a U-shape pH dependence. The binding was dissected to contributing binding-linked reactions and the intrinsic binding reaction affinity was calculated. This value is independent of pH and protonation reactions that occur simultaneously upon binding native CA and as demonstrated here, to metal substituted CA. Sulfonamide inhibitor binding to cancer-associated isozyme CAIX diminished in the order: Zn > Co > Hg > Cu > Cd > Mn > Ni. Energetic contribution of the inhibitor-metal coordination bond was determined for all above metals. The understanding of the principles of metal influence on ligand affinity and selectivity should help design new drugs targeting metalloenzymes.

Enhancing Chromatographic Analysis: A Novel Cryo-Enrichment Module for GC Analysis of Terpenes in Cannabis sativa.

This study presents the development and validation of a gas chromatography (GC) method using cryo-enrichment for the analysis of α-pinene and ß-caryophyllene in Cannabis sativa plant. Cryo-enrichment is a method that involves cooling part of the GC column to improve some aspect of chromatographic analysis. The aim of this study was to improve upon previous GC cryo-enrichment prototype and to create and validate a method for the analysis of these terpene compounds, which have received increasing attention for their medicinal properties. The improved cryo-enrichment device used in this study was built in-house and used a custom-built aluminum GC column holder cooled by a two-stage cryo-cooler. The validated method was found to be precise, accurate and sensitive, with good repeatability.

Cheap and easy human breath collection system for trace volatile organic compounds screening using thermal desorption - gas chromatography mass spectrometry.

•By analyzing the VOCs presents in our breath, we could identify if some components should not be present in our bodies, or their concentration is higher or lower than normal.•To collect breath samples for VOC analysis, we looked into the current available methodologies and, due to their high prices, tried to develop our own easy and cheap device. A simple single use Minigrip LDPE plastic bag was used in this work and its efficiency and performance were tested.•After breath collection, samples were analyzed using Thermal Desorption (TD) system, coupled with Gas Chromatography Mass Spectrometer (GC-MS).

Relation between thickness, crystallite size and magnetoresistance of nanostructured La1- x Sr x Mn y O3±Î´ films for magnetic field sensors.

In the present study the advantageous pulsed-injection metal organic chemical vapour deposition (PI-MOCVD) technique was used for the growth of nanostructured La1- x Sr x Mn y O3±Î´ (LSMO) films on ceramic Al2O3 substrates. The compositional, structural and magnetoresistive properties of the nanostructured manganite were changed by variation of the processing conditions: precursor solution concentration, supply frequency and number of supply sources during the PI-MOCVD growth process. The results showed that the thick (≈400 nm) nanostructured LSMO films, grown using an additional supply source of precursor solution in an exponentially decreasing manner, exhibit the highest magnetoresistance and the lowest magnetoresistance anisotropy. The possibility to use these films for the development of magnetic field sensors operating at room temperature is discussed.

Matrix solid-phase dispersion extraction of carbadox and olaquindox in feed followed by hydrophilic interaction ultra-high-pressure liquid chromatographic analysis.

A new method involving matrix solid-phase dispersion (MSPD) extraction and hydrophilic interaction ultra-high-pressure liquid chromatography (HILIC-UHPLC) with photodiode array detection was developed for the determination of carbadox and olaquindox in feed. Separation of carbadox and olaquindox was achieved within 1 min on the 1.7 microm Acquity UPLC BEH HILIC column by using isocratic elution with a mobile phase consisting of 10 mmol L(-1) ammonium acetate in acetonitrile-water (95:5, v/v) at a flow rate of 0.5 mL min(-1). Optimization of MSPD extraction parameters, such as type of solid sorbent and elution solvent were carried out. Optimal conditions selected for MSPD extraction were: 0.25 g of feed sample, 0.5 g of octadecylsilica as solid sorbent and 10 mL of acetonitrile-methanol (8:2, v/v) as eluting solvent. Both analytes provided average recoveries from spiked feed samples ranging from 89.1 to 98.4% with relative standard deviations less than 10%. Obtained performance characteristics are comparable to those achieved by liquid-liquid extraction-HPLC with the advantages of being simpler and significantly faster.

Speciation of Co(II), Co(III), and Cu(II) in ethylenediamine solutions by capillary electrophoresis.

A capillary electrophoretic (CE) method for the speciation of Co(II), Co(III), and Cu(II) in electroless copper-plating baths containing ethylenediamine (En) has been developed. The method is based on the selective pre-capillary derivatization of Co(II) with 1,10-phenanthroline (Phen) followed by CE separation of stable [CoPhen(3)](2+), [CoEn(3)](3+), and [CuEn(2)](2+) chelates. The proposed derivatization procedure protects Co(II) from oxidation by dissolved oxygen and enables rapid determination of all three metal species within a single run. The optimized separations were carried out in a fused silica capillary (57 cmx75-microm I.D.) filled with an ethylenediamine sulfate electrolyte (20 mmol L(-1) H(2)SO(4), pH 7.0 with En, applied voltage +30 kV) using direct UV detection at 214 nm. The detection limits for a signal-to-noise ratio of 3 and 10 s, hydrodynamic injections were 5x10(-6) mol L(-1) for Cu(II), 1x10(-6) mol L(-1) for Co(III), and 4x10(-7) mol L(-1) for Co(II). Application of the method to the speciation of Co(II), Co(III), and Cu(II) in copper-plating bath samples is also demonstrated.

Development of capillary electrophoresis for the determination of metal ions using mixed partial and complete complexation techniques.

A new capillary electrophoretic (CE) method was developed for the selective and sensitive determination of common metal ions. The proposed method is based on conventional CE separation of metal cations followed by complete complexation of separated analytes with 1,10-phenanthroline using the zone-passing technique. This approach combines both partial and complete complexation modes and, thus, enables rapid, selective, efficient separation together with sensitive direct UV detection of metal species. The optimal conditions for the separation and derivatization reaction were established by varying type of electrolyte, electrolyte pH, introduction time and concentration of 1,10-phenanthroline. The optimized separations were carried out in 50 mmol l(-1) glycolic acid electrolyte (pH 6.0 with imidazole) using direct UV detection at 254 nm. Five common metal cations (Fe2+, Co2+, Ni2+, Cu2+ and Zn2+) were separated in less than 4 min. The proposed system was applied to the determination of Fe(II) and Zn(II) in snow samples. The recovery tests established for snow samples were within the range 100+/-12%.