High-performance liquid chromatography assay for N-acetylcysteine in biological samples following derivatization with N-(1-pyrenyl)maleimide

N-Acetylcysteine is a thiol antioxidant with expanding clinical importance. A sensitive, rapid method for determining reduced N-acetylcysteine (NAC) concentration in biological samples has been developed which uses a modified reversed-phase high-performance liquid chromatography (HPLC) technique in conjunction with the derivatizing agent N-(1-pyrenyl)maleimide (NPM). The NAC-NPM adduct was analyzed by HPLC with fluorescence detection. The calibration curve for NAC was linear over the range 8–2500 nM and the coefficient of variation obtained for the within-run precision and the between-run precision for 0.5 mM NAC was 1.5% and 2.7%, respectively. Relative recovery of NAC from biological materials ranged between 86% and 96% and the limit of quantitation from biological samples was 32 nM. These results suggest practical advantages relative to other widely-accepted methods of NAC measurement.     

A methodology for simultaneous fluorogenic derivatization and boronate affinity enrichment of 3-nitrotyrosine-containing peptides

We synthesized and characterized a new tagging reagent, (3R,4S)-1-(4-(aminomethyl)phenylsulfonyl)pyrrolidine-3,4-diol (APPD), for the selective fluorogenic derivatization of 3-nitrotyrosine (3-NT) residues in peptides (after reduction to 3-aminotyrosine) and affinity enrichment. The synthetic 3-NT-containing peptide, FSAY(3-NO₂)LER, was employed as a model for method validation. Furthermore, this derivatization protocol was successfully tested for analysis of 3-NT-containing proteins exposed to peroxynitrite in the total protein lysate of cultured C2C12 cells. The quantitation of 3-NT content in samples was achieved through either fluorescence spectrometry or boronate affinity chromatography with detection by specific fluorescence (excitation and emission wavelengths of 360 and 510 nm, respectively); the respective limits of detection were 95 and 68 nM (19 and 13 pmol total amount) of 3-NT. Importantly, the derivatized peptides show a strong retention on a synthetic boronate affinity column, containing sulfonamide-phenylboronic acid, under mild chromatographic conditions, affording a route to separate the derivatized peptides from large amounts (milligrams) of nonderivatized peptides and to enrich them for fluorescent detection and mass spectrometry (MS) identification. Tandem MS analysis identified chemical structures of peptide 3-NT fluorescent derivatives and revealed that the fluorescent derivatives undergo efficient backbone fragmentations, permitting sequence-specific identification of protein nitration at low concentrations of 3-NT in complex protein mixtures.     

Development and validation of a new gas flame ionization detector method for the determination of finasteride in tablets

A simple, rapid, and sensitive method based on gas chromatography with flame ionization detection is described for the determination of finasteride in tablets. The method is based on the derivatization of finasteride with N,O-bis(trimethylsilyl)trifluoroacetamide-1% trimethylchlorosilane at 60 degrees C for 30 min. The method was validated for specificity, linearity, precision, accuracy, robustness, and limit of quantification. The degree of linearity of the calibration curves, the percentage recoveries of finasteride, and the limit of detection (LOD) and limit of quantification (LOQ) for the gas chromatographic method were determined. The assay was linear over the concentration range of 10 to 50 microg ml(-1) (R approximately 0.999). LOQ and LOD (signal/noise ratio = 10) were found to be 10 and 2 microg ml(-1), respectively. The method was found to be simple, specific, precise, accurate, and reproducible. All of the validation parameters were within the acceptance range. The developed method was applied successfully to estimate the amount of finasteride in tablets. The results were compared statistically with those obtained by the official method using t and F tests. There was no significant difference between the two methods with respect to mean values and standard deviations at the 95% confidence level.     

Survey of aliphatic glucosinolates in Sicilian wild and cultivated Brassicaceae

In the frame of the activities carried out to exploit Sicilian local cultivars of brassicas, we focused our attention on some of the potential health compounds of various local cruciferous crops. These compounds are of interest to improve the quality of the produce with the aim to develop new cultivars capable of providing functional foods able to prevent disease. In this context, we surveyed for the presence of specific glucosinolates in local cultivars of broccoli, cauliflower, kale, and in some wild species widespread in Sicily, using as control various commercial cultivars. Glucosinolate composition varied extensively among species and crops of the same species, such as cauliflower, broccoli and kale. Cultivar variation for glucosinolate profile was also observed for some crops. For example, Sicilian cultivars of cauliflower possessing colored curds displayed a high content of glucosinolates, glucoraphanin in particular, compared to white curd commercial cultivars. Also some wild species had a high content of other glucosinolates.     

Determination of the absolute configuration of the glucosinolate methyl sulfoxide group reveals a stereospecific biosynthesis of the side chain

Glucosinolates are plant metabolites containing an anionic nitrogeneous thioglucosidic core structure and a structurally diverse amino acid-derived side chain, which after hydrolysis by thioglucohydrolases (myrosinases) afford biological active degradation products such as nitriles and isothiocyanates. Structural diversity in glucosinolates is partially due to enzymatic modifications occurring on the preformed core structure, like the recently described oxidation of sulfides to sulfoxides catalyzed by a flavin monooxygenase identified in Arabidopsis thaliana. The enzyme product, 4-methylsulfinylbutylglucosinolate, bears a chiral sulfoxide group in its side chain. We have analyzed the epimeric purity of 4-methylsulfinylbutylglucosinolate by NMR methods using a chiral lanthanide shift reagent. The absolute configuration of the sulfoxide group has been established by comparing the ¹H NMR spectra of the two sulfoximine diastereomers of natural 4-methylsulfinylbutylglucosinolate. According to our data, 4-methylsulfinylbutylglucosinolate isolated from broccoli and A. thaliana is a pure epimer and its sulfoxide group has the R_S configuration. The product of the A. thaliana flavin monooxygenase has these same properties demonstrating that the enzyme is stereospecific and supporting its involvement in glucosinolate side chain formation.     

A dye-binding assay for measurement of the binding of Cu(II) to proteins

We analysed the theory of the coupled equilibria between a metal ion, a metal ion-binding dye and a metal ion-binding protein in order to develop a procedure for estimating the apparent affinity constant of a metal ion:protein complex. This can be done by analysing from measurements of the change in the concentration of the metal ion:dye complex with variation in the concentration of either the metal ion or the protein. Using experimentally determined values for the affinity constant of Cu(II) for the dye, 2-(5-bromo-2-pyridylaxo)-5-(N-propyl-N-sulfopropylamino) aniline (5-Br-PSAA), this procedure was used to estimate the apparent affinity constants for formation of Cu(II):transthyretin, yielding values which were in agreement with literature values. An apparent affinity constant for Cu(II) binding to α-synuclein of ∼1 × 10⁹ M⁻¹ was obtained from measurements of tyrosine fluorescence quenching by Cu(II). This value was in good agreement with that obtained using 5-Br-PSAA. Our analysis and data therefore show that measurement of changes in the equilibria between Cu(II) and 5-Br-PSAA by Cu(II)-binding proteins provides a general procedure for estimating the affinities of proteins for Cu(II).     

Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates

In the present study, soluble, functionally-active, recombinant human mitochondrial intermediate peptidase (hMIP), a mitochondrial metalloendoprotease, was expressed in a prokaryotic system. The hMIP fusion protein, with a poly-His-tag (6× His), was obtained by cloning the coding region of hMIP cDNA into the pET-28a expression vector, which was then used to transform Escherichia coli BL21 (DE3) pLysS. After isolation and purification of the fusion protein by affinity chromatography using Ni-Sepharose resin, the protein was purified further using ion exchange chromatography with a Hi-trap resource Q column. The recombinant hMIP was characterized by Western blotting using three distinct antibodies, circular dichroism, and enzymatic assays that used the first FRET substrates developed for MIP and a series of protease inhibitors. The successful expression of enzymatically-active hMIP in addition to the FRET substrates will contribute greatly to the determination of substrate specificity of this protease and to the development of specific inhibitors that are essential for a better understanding of the role of this protease in mitochondrial functioning.     

A sensitive mass spectrometry method for simultaneous quantification of DNA methylation and hydroxymethylation levels in biological samples

The recent discovery of 5-hydroxymethyl-cytosine (5hmC) in embryonic stem cells and postmitotic neurons has triggered the need for quantitative measurements of both 5-methyl-cytosine (5mC) and 5hmC in the same sample. We have developed a method using liquid chromatography electrospray ionization tandem mass spectrometry with multiple reaction monitoring (LC–ESI–MS/MS–MRM) to simultaneously measure levels of 5mC and 5hmC in digested genomic DNA. This method is fast, robust, and accurate, and it is more sensitive than the current 5hmC quantitation methods such as end labeling with thin layer chromatography and radiolabeling by glycosylation. Only 50 ng of digested genomic DNA is required to measure the presence of 0.1% 5hmC in DNA from mouse embryonic stem cells. Using this procedure, we show that human induced pluripotent stem cells exhibit a dramatic increase in 5mC and 5hmC levels compared with parental fibroblast cells, suggesting a dynamic regulation of DNA methylation and hydroxymethylation during cellular reprogramming.     

Soil core method for direct simultaneous determination of N2 and N2O emissions from forest soils

In order to quantify N₂-emissions from a spruce and a beech site at the Höglwald Forest, a new measuring system was developed, that allowed simultaneous, direct determination of N₂- and N₂O-emission with high accuracy (detection limit approx. 10 g N m^–2 h^–1 for N₂ and <1 g for N₂O) using a gas-flow core method. This method requires exchange of the soil atmosphere with an artificial atmosphere, that differs only in that N₂ is substituted by He. The measuring system, the methodology of measurements and validation experiments are described in detail. Due to the huge heterogeneity of denitrification activity in different soil cores taken from our forest sites, no general trends of N₂ and N₂O production in relation to soil moisture and temperature could be demonstrated. Based on reasonable number of measurements, this work gives for the first time an estimate of the magnitude of N₂-losses from temperate forest soils. Both the magnitude of N₂-emissions (spruce: 7.2±0.7 kg N₂-N ha^–1 yr^–1; beech: 12.4±3.1 kg N₂-N ha^–1 yr^–1), as well as the N₂O–N₂ ratio (spruce: 0.136±0.04; beech: 0.52±0.19) were significantly higher for soils from the beech sites as compared to soils from the spruce site. The results suggests that N₂-emissions from N-saturated forest soils, still receiving high loads of atmospheric N-deposition, are approx. 30% of atmospheric N-input at the spruce site, and approx. 50% at the beech site. Our results demonstrate that losses of nitrogen in the form of N₂ cannot be neglected in the context of calculating N-balances for given forest sites.     

Recovery of nitrogen fixation after short-term flooding of the nodulated root system of soybean

Nitrogen fixation of terrestrial legumes is strongly and rapidly diminished under flooding. Although recovery is possible with the formation of aerenchyma, information is scarce regarding recovery after draining following short-term flooding, before the appearance of morphological adaptations. This study used soybean (Glycine max) plants nodulated with Bradyrhizobium elkanii to determine xylem sap glutamine as an indication of nitrogen fixation activity during recovery from different periods of flooding. Xylem glutamine levels showed rapid recovery (within 90 min) following periods of flooding up to 4 h. Recovery was progressively slower after longer periods of flooding. After 48 h flooding very little recovery could be observed within the first 120 min after draining but recovery was possible within 48 h. Consistent with the changes in xylem glutamine, direct measurements of apparent nitrogenase activity carried out immediately on draining revealed rapid recovery after flooding for 1 h and slow recovery following 48 h of flooding. In the latter case, nitrogenase activity largely recovered 24 h after draining. Experiments with ¹⁵N₂ incorporation into amino acids exported in the xylem sap revealed that glutamine was by far the most highly labelled amino acid in sap collected over the first 30 min of exposure to the isotope. This is conclusive evidence that xylem sap glutamine is an immediate product of N₂ fixation and export. The changes in xylem sap glutamine seen on flooding (decline) and after draining (recovery) can therefore be attributed to changes in nitrogenase activity. The data show that xylem sap glutamine is a useful means for assessing changes in nitrogenase activity, especially when the root system is submersed in water and activity cannot be measured directly.     

Design of experiments: an efficient strategy to identify factors influencing extraction and derivatization of Arabidopsis thaliana samples in metabolomic studies with gas chromatography/mass spectrometry

The usual aim in metabolomic studies is to quantify the entire metabolome of each of a series of biological samples. To do this for complex biological matrices, e.g., plant tissues, efficient and reproducible extraction protocols must be developed. However, derivatization protocols must also be developed if GC/MS (one of the mostly widely used analytical methods for metabolomics) is involved. The aim of this study was to investigate how different chemical and physical factors (extraction solvent, derivatization reagents, and temperature) affect the extraction and derivatization of the metabolome from leaves of the plant Arabidopsis thaliana. Using design of experiment procedures, variation was systematically introduced, and the effects of this variation were analyzed using regression models. The results show that this approach allows a reliable protocol for metabolomic analysis of Arabidopsis to be determined with a relatively limited number of experiments. Following two different investigations an extraction and derivatization protocol was chosen. Further, the reproducibility of the analysis of 66 endogenous compounds was investigated, and it was shown that both hydrophilic and lipophilic compounds were detected with high reproducibility.     

Probing electron transfer reactions between two azurins from Alcaligenes xylosoxidans GIFU 1051 with optically active Ru complexes as molecular recognition probes: Importance of the 43rd residue

Electron transfer reactions between optically-active Ru^II/III complexes incorporating (S)-/(R)-amino acids, and the two azurins, azurin-1 (az-1Cu) and azurin-2 (az-2Cu) isolated from Alcaligenes xylosoxidans GIFU 1051, have been studied to probe molecular recognition sites on the two azurins. The Ru^II/III complexes are K[Ru^II(L)(bpy)] and [Ru^III(L)(bpy)], and have a tripodal ligand (L) derived from the (S)-/(R)-amino acids, which are in turn exchanged for other functional substituent groups, such as (S)-/(R)-phenylalanine, -leucine, -valine, -alanine, and -glutamic acid (L = (S)-/(R)-BCMPA, -BCMLE, -BCMVA, -BCMAL, and -BCMGA). In the oxidation reaction of az-1Cu^I promoted by the Ru^III complexes, the kinetic parameters exhibited enantio- and stereo-selectivities, while the same reaction of az-2Cu^I was less enantio- and stereo-selective. These differences suggest that the processes of formation of the activated states are different for the two azurins. On the other hand, such a difference has not been observed for az-1 and az-2 with respect to the reduction reactions promoted by both azurins Cu^II by the Ru^II complexes within the experimental error. This suggests that the neutrality of the Ru complexes is important for precise molecular recognition of azurins. His117 has been proposed as the electron transfer site. The local structures in the vicinity of the His117 side chain in the two azurins, are essentially identical with the exception of the 43rd residue, Val43 and Ala43 for az-1 and az-2, respectively. Electron transfer reactions between Ru^III complexes and a mutant azurin, V43A-az-1, were also carried out. Interestingly, the activation parameters estimated were very similar to those of az-2, indicating that the 43rd residue acts as the electron transfer site in azurins and provides rationalization for the different mechanisms of az-1 and az-2 in redox reactions.     

A pi-helix switch selective for porphyrin deprotonation and product release in human ferrochelatase

Ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) is the terminal enzyme in heme biosynthesis and catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme IX (heme). Due to the many critical roles of heme, synthesis of heme is required by the vast majority of organisms. Despite significant investigation of both the microbial and eukaryotic enzyme, details of metal chelation remain unidentified. Here we present the first structure of the wild-type human enzyme, a lead-inhibited intermediate of the wild-type enzyme with bound metallated porphyrin macrocycle, the product bound form of the enzyme, and a higher resolution model for the substrate-bound form of the E343K variant. These data paint a picture of an enzyme that undergoes significant changes in secondary structure during the catalytic cycle. The role that these structural alterations play in overall catalysis and potential protein-protein interactions with other proteins, as well as the possible molecular basis for these changes, is discussed. The atomic details and structural rearrangements presented herein significantly advance our understanding of the substrate binding mode of ferrochelatase and reveal new conformational changes in a structurally conserved pi-helix that is predicted to have a central role in product release.     

"Distant spin trapping": a method for expanding the availability of spin trapping measurements

The technique of spin trapping is used to study a wide range of free radicals in various systems, including those generated in vitro and in vivo. But unfortunately, EPR spectrometers are not always immediately accessible at the site of experimentation, and therefore it is important to find a method that can preserve a radical adduct over longer periods of time. We describe here an alternative method in which the samples can be frozen and transported for EPR measurements at another site. Various spin adducts of DEPMPO were frozen and measured at 0 degrees C at various intervals after freezing to determine their stability in the frozen state. The radical adducts were generated by established methods and stored at two different temperatures; -196 degrees C (liquid nitrogen) and -80 degrees C (dry ice). The experiments were carried out in an aqueous solution with and without a model of reducing environment (2 mM ascorbate). The results indicate that it is feasible to store and transport spin adducts for subsequent analysis. We conclude that this approach, which we term "distant spin trapping", makes it feasible to transport samples to another site for EPR measurements. This should significantly expand the ability to use spin trapping in biology and medicine.     

Puroindolines form ion channels in biological membranes

Wheat seeds contain different lipid binding proteins that are low molecular mass, basic and cystine-rich proteins. Among them, the recently characterized puroindolines have been shown to inhibit the growth of fungi in vitro and to enhance the fungal resistance of plants. Experimental data, using lipid vesicles, suggest that this antimicrobial activity is related to interactions with cellular membranes, but the underlying mechanisms are still unknown. This paper shows that extracellular application of puroindolines on voltage-clamped Xenopus laevis oocytes induced membrane permeabilization. Electrophysiological experiments, on oocytes and artificial planar lipid bilayers, suggest the formation, modulated by voltage, of cation channels with the following selectivity: Cs(+) > K(+) > Na(+) > Li(+) > choline = TEA. Furthermore, this channel activity was prevented by addition of Ca(2+) ions in the medium. Puroindolines were also able to decrease the long-term oocyte viability in a voltage-dependent manner. Taken together, these results indicate that channel formation is one of the mechanisms by which puroindolines exert their antimicrobial activity. Modulation of channel formation by voltage, Ca(2+), and lipids could introduce some selectivity in the action of puroindolines on natural membranes.     

Kinetics and mechanism of large rate enhancement in an acidic aqueous cleavage of the tertiary amide bond of N-(2-methoxyphenyl)-N'-morpholinophthalamide (1)

The rate of conversion of 1 to N-(2-methoxyphenyl)phthalimide (2) within [HCl] range 5.0 × 10⁻³-1.0 M at 1.0 M ionic strength (by NaCl) reveals the presence of both uncatalyzed and specific acid-catalyzed kinetic terms in the rate law. Intramolecular carboxamide group-assisted cleavage of amide bond of 1 reveals rate enhancement of much larger than 10⁶-fold compared to the expected rate of analogous intermolecular reaction.     

Synthesis and coordination behaviors of 14-membered tetraaza macrocyclic copper(II) complexes bearing two N-propionic acid or N-propionic methyl ester groups

A di-N-functionalized 14-membered tetraaza macrocycle, [H₄L³](ClO₄)₂ (L³ = 1,8-bis(2-carboxyethyl)-3,5,7,7,10,12,14,14-octamethyl-1,4,8,11-tetraazacyclotetradecane), has been synthesized by acid hydrolysis of 1,8-bis(2-cyanoethyl)-3,5,7,7,10,12,14,14-octamethyl-1,4,8,11-tetraazacyclotetradecane (L²). The copper(II) complexes [CuL²](ClO₄)₂ and [Cu(H₂L³)](ClO₄)₂ were prepared and characterized. The complex [Cu(H₂L³)]²⁺ readily reacts with methanol to yield [CuL⁴]²⁺ (L⁴ = 1,8-bis(2-carbomethoxyethyl)-3,5,7,7,10,12,14,14-octamethyl-1,4,8,11-tetraazacyclotetradecane). The N-CH₂CH₂COOH groups of [Cu(H₂L³)](ClO₄)₂ are not coordinated to the metal ion in the solid state but are involved in coordination in various non-aqueous solvents or in aqueous solutions of pH ? 1.0. Interestingly, [CuL⁴](ClO₄)₂ exists as two stable structural isomers, 1 (the pendant ester groups are not involved in coordination) and 2 (one of the two ester groups is coordinated to the metal ion), in the solid state; the two isomers can be prepared selectively by controlling ionic strength of a methanol solution of the complex. Crystal structures and coordination behaviors of the two isomers are described. The di-N-cyanoethylated macrocyclic complex [CuL²](ClO₄)₂ is rapidly decomposed in 0.1 M NaOH solution even at room temperature. On the other hand, [Cu(H₂L³)](ClO₄)₂ and [CuL⁴](ClO₄)₂ are quite inert against decomposition under similar basic conditions. In acidic or basic aqueous solutions, [CuL⁴]²⁺ is hydrolyzed to [Cu(H₂L³)]²⁺ or [CuL³].     

Sequence variation in CYP51A from the Y strain of Trypanosoma cruzi alters its sensitivity to inhibition

CYP51 (sterol 14α-demethylase) is an efficient target for clinical and agricultural antifungals and an emerging target for treatment of Chagas disease, the infection that is caused by multiple strains of a protozoan pathogen Trypanosoma cruzi. Here, we analyze CYP51A from the Y strain T. cruzi. In this protein, proline 355, a residue highly conserved across the CYP51 family, is replaced with serine. The purified enzyme retains its catalytic activity, yet has been found less susceptible to inhibition. These biochemical data are consistent with cellular experiments, both in insect and human stages of the pathogen. Comparative structural analysis of CYP51 complexes with VNI and two derivatives suggests that broad-spectrum CYP51 inhibitors are likely to be preferable as antichagasic drug candidates.     

Synthesis of [I]iodoDPA-713: A new probe for imaging inflammation

[¹²⁵I]IodoDPA-713 [¹²⁵I]1, which targets the translocator protein (TSPO, 18 kDa), was synthesized in seven steps from methyl-4-methoxybenzoate as a tool for quantification of inflammation in preclinical models. Preliminary in vitro autoradiography and in vivo small animal imaging were performed using [¹²⁵I]1 in a neurotoxicant-treated rat and in a murine model of lung inflammation, respectively. The radiochemical yield of [¹²⁵I]1 was 44 ± 6% with a specific radioactivity of 51.8 GBq/μmol (1400 mCi/μmol) and >99% radiochemical purity. Preliminary studies showed that [¹²⁵I]1 demonstrated increased specific binding to TSPO in a neurotoxicant-treated rat and increased radiopharmaceutical uptake in the lungs of an experimental inflammation model of lung inflammation. Compound [¹²⁵I]1 is a new, convenient probe for preclinical studies of TSPO activity.