Separation/Preconcentration Methods for the Determination of Aluminum in Dialysate Solution and Scalp Hair Samples of Kidney Failure Patients

A new method is reported for the separation of aluminum ions (Al(3+)) from interfering cations in pharmaceutical and biological samples through solid-phase extraction (SPE) using 2-methyl-8-hydroxyquinoline (8-hydroxyquinaldine) on activated silica. While separated Al(3+) was preconcentrated by cloud point extraction (CPE) using 3,5,7,2'-4'-pentahydroxyflavone (morin) as complexing reagent, the resulting complex was entrapped in nonionic surfactant (Triton X-114) as prior step to its determination by spectrofluorimetry (SPF). The validity of separation/preconcentration of Al(3+) was checked by certified reference material of human hair and standard addition method. The chemical variables affecting the analytical performance of the separation/preconcentration methods were studied and optimized. The enrichment factor and detection limit of Al(3+) for the preconcentration of 10?ml of dialysate solution and acid-digested samples of scalp hair samples were found to be 25 and 0.34?μg/L, respectively. The relative standard deviation for six replicates of standard containing 20?μg/L of Al(3+) was <10%. In all DS, the concentration of Al was >10?μg/L. The level of Al in scalp hair samples of kidney failure patients was higher than healthy controls.     

Simultaneous determination of Fe(III) and Al(III) by first-derivative spectrophotometry and partial least-squares (PLS-2) method – Application to post-haemodialysis fluids

Derivative spectrophotometry (graphical method) and partial least-squares regression (numerical method) methods were developed for the spectrophotometric multi-component analysis of post-haemodialysis fluids and synthetic mixtures containing Al(III) and Fe(III) without any chemical separation. The complexes of these metal ions with chrome azurol S were formed immediately at pH 5.5 and were stable for at least 3h. The graphical method is based on the use of first-derivative spectra for evaluation because working wavelength determination was more precise and spectral overlap was less than in the ordinary spectra. Two wavelengths at which the complexes exhibited maximum absorption values for Fe(III) and Al(III) were selected as analytical wavelengths, i.e., 675 and 623.5nm, respectively. Lambert-Beer's law is obeyed between 0.0896-8.064mug/mL Fe(III) and 0.054-0.486mug/mL Al(III). Limits of detection for Fe(III) and Al(III) were 0.056 and 0.044mug/mL, respectively. The reproducibility, expressed as variation coefficients, for two sets of 10 standard mixtures containing 3.584mug/mL Fe(III) and 0.27mug/mL Al(III) were 1.9% and 2% for iron and aluminium, respectively. In the numerical method, a training set was randomly prepared by using 14 samples. The concentration of each component has been varied in the linear range of the analytical signal. The spectral regions between 510 and 720nm were selected for the analysis of the binary mixture of Fe(III)/Al(III). The proposed methods were validated by using synthetic binary mixtures and applied to the simultaneous determination of Fe(III) and Al(III) in post-haemodialysis samples. The obtained results were compared with each other; in general, both multi-component methods gave rise to similar recovery results for laboratory-prepared mixtures and real samples.     

Aluminum compounds enhance lipid peroxidation in liposomes: insight into cellular damage caused by oxidative stress

Aluminum (Al) has been proposed as one of the critical environmental factors responsible for several neurodegenerative diseases such as Alzheimer's disease. However, the suggested mechanism involving the contribution of reactive oxygen species still remains controversial. We have first attempted to identify Al compounds either in its ionic or complexed forms that cause oxidative stress in biological systems. For this purpose, we examined the effect of inorganic Fe(2+)- and organic radical initiator (2,2'-azobis (2-amidinopopane) hydrochloride; AAPH)-induced lipid peroxidation by using aluminum (Al(3+)) nitrate and tris(maltolato)aluminum(III) complex (ALM) with respect to molecular oxygen (O(2)) consumption and membrane fluidity change in liposomes as biological membrane models. The following important results were obtained: (1) ALM enhanced the lipid peroxidation induced by Fe(2+) and AAPH in phosphatidylcholine liposomes; this corresponded well with the promotion of O(2) uptake in the same liposomes, (2) Al(3+) increased both lipid peroxidation and O(2) consumption in phosphatidylserine liposomes in the presence of Fe(2+), and (3) both Al(3+) and ALM affected the membrane fluidity on the inner side. It has been concluded that ALM induces higher lipid peroxidation in liposomes than Al(3+); this finding will be useful to gain an insight into the role of Al in cellular damage in relation to oxidative stress.     

Interactions of aluminium(III) with glycerolphosphates and glycerophosphorylcholine

The complexation of aluminium(III) with glycerol-1-phosphate (G1P) and glycerol-2-phosphate (G2P) in aqueous solutions has been studied as a function of pH, by pH-potentiometry, 31P NMR spectroscopy and ESI mass spectrometry. Various mononuclear complexes (MLH(2)(3+), MLH(2+), ML(+), ML(2)H, ML(2)(-)) and polynuclear species (M(3)L(3)H(-1)(2+), M(3)L(2)H(-n)((n-5)-) with n=5, 6, 7, M(2)L(2)H(-1)(+) ) are formed in the system where the full protonated ligands are noted LH(2). NMR experiments clearly show that G1P and G2P already interact with Al(III) at pH 1. The potentiometric results are confirmed by ESI measurements and 31P NMR studies. No metal ion-induced deprotonation and coordination of the alcoholic-OH functions seem to occur during the complexation. The situation is very different for the glycerophosphorylcholine ligand (GPC identical with LH). Only the complex ML(3+) is formed in aqueous solution with a relatively low formation constant (K=5 at 37 degrees C). This species is clearly identified in 31P and 27Al NMR spectra. The complexation study as a function of the temperature allowed us to determine the thermodynamic parameters of the complex formation. The complexation is not governed by the reaction enthalpy that is found to be positive but by the entropy that is largely positive.     

Determination of 5-fluorouracil in environmental samples by solid-phase extraction and high-performance liquid chromatography with ultraviolet detection

5-Fluorouracil (5-FU) is one of the most widely used antineoplastic drugs. It can be therefore considered to be a model compound for the identification of exposure routes during preparation and administration of cytostatic agents, especially for nucleoside analogue drugs. In this study, an HPLC–UV method was validated for determination of 5-FU in wipe samples by direct analysis of the aqueous solutions and in air samples by using solid-phase extraction (SPE). When samples were pre-treated on styrene–divinylbenzene resin SPE columns, a 20-fold preconcentration of the analyte was achieved. As regards air samples, correlation coefficients were always higher than 0.998 and the limit of detection was assessed at 15 ng on filter. In order to verify the reliability of these procedures, 5-chlorouracil was used as internal standard. The procedure presented here has been applied to the environmental monitoring of occupational exposed subjects. The amount of 5-FU ranged from 0.043 to 0.23 μg/m³ in air samples and from 0.2 to 470.1 μg/dm² in wipe samples. 5-FU was also detected on the internal side of the gloves (0.07 to 3.77 μg/pair of gloves).     

Determination of polychlorinated biphenyls in small-size serum samples by solid-phase extraction followed by gas chromatography with micro-electron-capture detection

An new method for the determination of polychlorinated biphenyls (PCBs) in serum samples of up to 1 ml has been developed. The procedure consisted in the solid-phase extraction (SPE) of the analytes on an Oasis cartridge and the subsequent on-line elimination of the fat by directly dropping of the eluate from the SPE cartridge on a multilayer column placed below the cartridge. This configuration allowed minimising of the sample manipulation as well as the time, solvent and sorbent consumption (i.e. complete sample preparation can be accomplished in about 1 h with only 3 ml of toluene and 300 mg of silica). The SPE plus clean-up method developed showed a satisfactory performance for the analysis of PCBs in rat serum samples providing similar recoveries (i.e. range 73-128% for most of the congeners selected) at the different spiking levels investigated (1.25, 0.50 and 0.25 ng/ml). Detection limits using a microelectron capture detector were in the range 0.01-0.30 ng/ml of serum and the relative standard deviations of the complete method better than 18% irrespective of the PCB concentration. The validated method has been applied to the evaluation for the first time of the PCB levels in serum samples of up to 1 ml from individuals of an Egyptian Vulture colony in Spain.     

Preconcentration of pharmaceuticals residues in sediment samples using microwave assisted micellar extraction coupled with solid phase extraction and their determination by HPLC-UV

An analytical method combining microwave assisted micellar extraction (MAME) and solid phase extraction (SPE) has been developed to extract and preconcentrate a selected group of eight pharmaceutical compounds in sediment samples prior to their determination using liquid chromatography with an UV-DAD detector. A non-ionic surfactant, Polyoxyethylene 10 lauryl ether (POLE) was used for the MAME extraction and the different parameters for the optimization process were studied. Then, SPE was used to clean-up and preconcentrate the target analytes in the extract, prior to their determination using HPLC-UV. The method was applied to the determination of the selected pharmaceuticals compounds in several kinds of sediment samples with different characteristics. Relative recoveries for spiked sediment samples were over 70% and relative standard deviations (RSDs) were under 11% for all recoveries tested. Detection limits between 4 and 167ngg(-1) were obtained. The method was validated using Soxhlet extraction procedure.     

Linear scan voltammetric indirect determination of Al(III) by the catalytic cathodic response of norepinephrine at the hanging mercury drop electrode

The biological effects of aluminum (Al) have received much attention in recent years. Al is of basic relevance as concern with its reactivity and bioavailability. In this paper, the electrochemical behaviors of norepinephrine (NE) in the absence and presence of Al(III) at the hanging mercury drop electrode have been studied and applied to the practical analysis. Highly selective catalytic cathodic peak of NE is yielded by linear scan voltammetry (LSV) at -1.32 V (vs. SCE). A linear relationship holds between the cathodic peak current and the Al(III) concentration. It has been successfully applied to the determination of Al(III) in real waters and synthetic biological samples with satisfying results, which are in accordance with those obtained by ICP-AES method. The electrochemical properties and the mechanisms of the peaks in the presence and absence of Al(III) have been explored. The results show that they are irreversible adsorptive hydrogen catalytic waves. These studies not only enrich the methods of determining Al, but also lay foundations of further understanding of the mechanisms of neurodementia.     

Potential of a simple solid-phase extraction method coupled to analytical and bioanalytical methods for an improved determination of microcystins in algal samples

Artemia assays and protein phosphatase assays are commonly used for the screening of microcystins (MCs) in algal samples instead of the standard mouse toxicity assay. However, it has been shown that their results are often biased because of the matrix effects. To eliminate the possible interferences in the algal matrices, a new solid-phase extraction (SPE) method using silica gel as a sorbent was developed and evaluated. Results show that this SPE method could not only reduce the toxicity of the Microcystis samples towards brine shrimp by 50-80% but also eliminate 90-100% of the endogenous phosphatase activity from Spirulina and Chlorella samples, thus improving the determination of microcystins in algal samples using either of the two bioanalytical methods. The application of this SPE method as an off-line cleanup for high-performance liquid chromatography (HPLC) with UV detection is also described in this study. After SPE, the HPLC chromatograms of Microcystis samples have clear baselines that have no interferences with the analyte peaks.     

Determination of trace amounts of total dissolved cationic aluminium species in environmental samples by solid phase extraction using nanometer-sized titanium dioxide and atomic spectrometry techniques

Nanometer-sized titanium dioxide was used as a solid-phase extractant for the separation and preconcentration of trace amounts of Al(III) prior to its determination by electrothermal atomic absorption spectrometry (ET AAS) and inductively coupled plasma optical emission spectrometry (ICP OES). The optimal conditions for the proposed solid phase extraction (SPE; 50 mg TiO₂, 10 min extraction time, pH 6.0, HCl and HNO₃ as eluents) and ET AAS measurement (1500 °C pyrolysis and 2600 °C atomization temperatures, Mg(NO₃)₂ as matrix modifier) were obtained. The adsorption capacity of TiO₂ was 4.1 mg Al g⁻¹ TiO₂. Two modes of the proposed procedure were compared, (I) batch and elution mode with the elution of Al from TiO₂ phase by nitric or hydrochloric acid, and (II) batch and slurry mode (without elution) with the direct TiO₂ phase-slurry sampling. Finally, the batch and slurry mode of nanometer-sized TiO₂ SPE with slurry ET AAS detection and quantification was preferred and used for the determination of trace amounts of total dissolved cationic Al species in synthetic and natural water samples. The method accuracy was checked by the analysis of lake water CRM TMDA-61 and by the technique of analyte addition (sample spiking). Under the optimal conditions, the calibration curve for batch and slurry TiO₂ SPE with a 10-fold preconcentration was linear up to 40 μg L⁻¹ Al. The limit of detection (LOD) and the limit of quantification (LOQ) was 0.11 μg L⁻¹ Al and 0.35 μg L⁻¹ Al, respectively, with a preconcentration factor of 20 and a relative standard deviation (RSD) lower than 5%.     

Role of the choline exchanger in Na(+)-independent Mg(2+) efflux from rat erythrocytes

Two types of Na(+)-independent Mg(2+) efflux exist in erythrocytes: (1) Mg(2+) efflux in sucrose medium and (2) Mg(2+) efflux in high Cl(-) media such as KCl-, LiCl- or choline Cl-medium. The mechanism of Na(+)-independent Mg(2+) efflux in choline Cl medium was investigated in this study. Non-selective transport by the following transport mechanisms has been excluded: K(+),Cl(-)- and Na(+),K(+),Cl(-)-symport, Na(+)/H(+)-, Na(+)/Mg(2+)-, Na(+)/Ca(2+)- and K(+)(Na(+))/H(+) antiport, Ca(2+)-activated K(+) channel and Mg(2+) leak flux. We suggest that, in choline Cl medium, Na(+)-independent Mg(2+) efflux can be performed by non-selective transport via the choline exchanger. This was supported through inhibition of Mg(2+) efflux by hemicholinum-3 (HC-3), dodecyltrimethylammonium bromide (DoTMA) and cinchona alkaloids, which are inhibitors of the choline exchanger. Increasing concentrations of HC-3 inhibited the efflux of choline and efflux of Mg(2+) to the same degree. The K(d) value for inhibition of [(14)C]choline efflux and for inhibition of Mg(2+) efflux by HC-3 were the same within the experimental error. Inhibition of choline efflux and of Mg(2+) efflux in choline medium occurred as follows: quinine>cinchonine>HC-3>DoTMA. Mg(2+) efflux was reduced to the same degree by these inhibitors as was the [(14)C]choline efflux.     

MCX based solid phase extraction combined with liquid chromatography tandem mass spectrometry for the simultaneous determination of 31 endocrine-disrupting compounds in surface water of Shanghai

A novel analytical method employing MCX (mixed-mode cationic exchange) based solid phase extraction (SPE) coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed to detect 31 endocrine-disrupting compounds (EDCs) in surface water samples simultaneously. The target EDCs belong to five classes, including seven estrogens, eight androgens, six progesterones, five adrenocortical hormones and five industrial compounds. In order to simultaneously concentrate the target EDCs and eliminate matrix interferences in the water samples, MCX SPE cartridges were employed for SPE, and then followed by a simple and highly efficient three-step sequential elution procedure. Two electrospray ionization (ESI) detection modes, positive (ESI+) and (ESI-), were optimized for HPLC-MS/MS analysis to obtain the highest sensitivity for all the EDCs. The limits of detection (LODs) were 0.02-1.9 ng L(-1), which are lower than or comparable to these reported in references. Wide linear ranges (LOD-100 ng L(-1) for ESI+ mode, and LOD-200 ng L(-1) for ESI- mode) were obtained with determination coefficients (R(2)) higher than 0.99 for all the compounds. With five internal standards, good recoveries (84.4-103.0%) of all the target compounds were obtained in selected surface water samples. The developed method was successfully applied to investigate the EDCs occurrence in the surface water of Shanghai by analyzing surface water samples from 11 sites. The results showed that nearly all the target compounds (30 in 31) were present in the surface water samples of Shanghai, of which three industrial compounds (4-t-OP, BPA, and BPF) showed the highest concentrations (median concentrations were 11.88-23.50 ng L(-1)), suggesting that industrial compounds were the dominating EDCs in the surface water of Shanghai, and much more attention should be paid on these compounds. Our present research demonstrated that SPE with MCX cartridges combined with HPLC-MS/MS was convenient, efficient and reliable for multiclass analysis of EDCs in surface water.     

Radial secondary electron dose profiles and biological effects in light-ion beams based on analytical and Monte Carlo calculations using distorted wave cross sections

To speed up dose calculation, an analytical pencil-beam method has been developed to calculate the mean radial dose distributions due to secondary electrons that are set in motion by light ions in water. For comparison, radial dose profiles calculated using a Monte Carlo technique have also been determined. An accurate comparison of the resulting radial dose profiles of the Bragg peak for (1)H(+), (4)He(2+) and (6)Li(3+) ions has been performed. The double differential cross sections for secondary electron production were calculated using the continuous distorted wave-eikonal initial state method (CDW-EIS). For the secondary electrons that are generated, the radial dose distribution for the analytical case is based on the generalized Gaussian pencil-beam method and the central axis depth-dose distributions are calculated using the Monte Carlo code PENELOPE. In the Monte Carlo case, the PENELOPE code was used to calculate the whole radial dose profile based on CDW data. The present pencil-beam and Monte Carlo calculations agree well at all radii. A radial dose profile that is shallower at small radii and steeper at large radii than the conventional 1/r(2) is clearly seen with both the Monte Carlo and pencil-beam methods. As expected, since the projectile velocities are the same, the dose profiles of Bragg-peak ions of 0.5 MeV (1)H(+), 2 MeV (4)He(2+) and 3 MeV (6)Li(3+) are almost the same, with about 30% more delta electrons in the sub keV range from (4)He(2+)and (6)Li(3+) compared to (1)H(+). A similar behavior is also seen for 1 MeV (1)H(+), 4 MeV (4)He(2+) and 6 MeV (6)Li(3+), all classically expected to have the same secondary electron cross sections. The results are promising and indicate a fast and accurate way of calculating the mean radial dose profile.     

Juvenile hormone biosynthesis in the fall armyworm, Spodoptera frugiperda (Lepidoptera, Noctuidae)

The in vitro production of juvenile hormones (JH) was investigated by using corpora allata (CA) of larvae and corpora cardiaca-corpora allata (CC-CA) complexes of adult females of the fall armyworm Spodoptera frugiperda. In female moths, JH release was high compared to that in 5th and 6th instar larvae. Concentrations of 0.11-0.12 mM methionine, 180-200 mM Na(+), 5.8-8.3 mM K(+), 10-50 mM Ca(2+) and a pH range of 5.7-6.3 yielded optimal incorporation of L-[methyl-(3)H] methionine in vitro by CC-CA complexes. The highest hourly incorporation occurred during a 9-h incubation period following a 1.5-h lag-phase. JH release from CC-CA complexes of adult females was shown to be age-dependent with a peak value on day 2 (approx. 4 pmol h(-1) CA(-1)). By a combination of reversed phase (RP)- and normal phase (NP)-high performance liquid chromatography (HPLC), two major labelled products released by the complex were separated. One compound co-migrated with chemically synthesized JH II diol, the second compound with JH III diol. Only traces of JH II and III could be detected in some samples. Gland extracts also contained both the major radiolabelled products. Double labelling experiments using [3H]methionine and [14C]acetate confirmed their de novo synthesis in CC-CA complexes of female moths. The nature of chemically synthesized reference JH III diol was proved by LC-MS (ESI mass spectrometry) and 1H-NMR (nuclear magnetic resonance spectroscopy).     

Selective Fe2+-catalyzed oxidative cleavage of gastric H+,K+-ATPase: implications for the energy transduction mechanism of P-type cation pumps

In the presence of ascorbate/H(2)O(2), Fe(2+) ions or the ATP-Fe(2+) complex catalyze selective cleavage of the alpha subunit of gastric H(+),K(+)-ATPase. The electrophoretic mobilities of the fragments and dependence of the cleavage patterns on E(1) and E(2) conformational states are essentially identical to those described previously for renal Na(+),K(+)-ATPase. The cleavage pattern of H(+),K(+)-ATPase by Fe(2+) ions is consistent with the existence of two Fe(2+) sites: site 1 within highly conserved sequences in the P and A domains, and site 2 at the cytoplasmic entrance to trans-membrane segments M3 and M1. The change in the pattern of cleavage catalyzed by Fe(2+) or the ATP-Fe(2+) complex induced by different ligands provides evidence for large conformational movements of the N, P, and A cytoplasmic domains of the enzyme. The results are consistent with the Ca(2+)-ATPase crystal structure (Protein Data Bank identification code; Toyoshima, C., Nakasako, M., Nomura, H., and Ogawa, H. (2000) Nature 405, 647-655), an E(1)Ca(2+) conformation, and a theoretical model of Ca(2+)-ATPase in an E(2) conformation (Protein Data Bank identification code ). Thus, it can be presumed that the movements of N, P, and A cytoplasmic domains, associated with the E(1) <--> E(2) transitions, are similar in all P-type ATPases. Fe(2+)-catalyzed cleavage patterns also reveal sequences involved in phosphate, Mg(2+), and ATP binding, which have not yet been shown in crystal structures, as well as changes which occur in E(1) <--> E(2) transitions, and subconformations induced by H(+),K(+)-ATPase-specific ligands such as SCH28080.     

A fluorimetric method for the determination of trace pentachlorophenol, based on its inhibitory effect on the redox reaction between the improved Fenton reagent and rhodamine B.

A sensitive fluorimetric method is presented and discussed for the determination of pentachlorophenol in aqueous solutions. This method is based on the inhibitory effect of pentachlorophenol on the reaction of conventional Fenton [Fe(III) + H(2)O(2)] reagent with rhodamine B in the medium of perchloric acid, which results in the fluorescence quenching of rhodamine B. It was further found that the sensitivity for the determination was improved significantly when the molecular ligand EDTA was added. This improved system was therefore presented for the determination of pentachlorophenol. The characteristics of the excitation and emission spectra, optimization of the experimental conditions, the stability of the system and the influence of foreign matter have all been investigated. Under optimal conditions, the linear range for the determination of pentachlorophenol is 12-480 ng/mL with a 3sigma limit of detection of 0.96 ng/mL. Compared with the conventional Fenton system, the improved system shows obvious advantages in both sensitivity and selectivity. By combination with the pretreatment of samples using ion exchange resins and XDA-1 absorption resin, the improved Fenton method was used for the first time for the determination of pentachlorophenol in synthetic samples and natural water samples, and satisfactory results, in agreement with those of the HPLC method, were achieved. The possible mechanism of the reactions has also been discussed. Copyright (c) 2007 John Wiley & Sons, Ltd.     

Synthesis of feralex a novel aluminum/iron chelating compound.

There is a need for in vivo applicable Fe(3+) and Al(3+) chelation compounds for use as medicines to treat toxic overload conditions of these ions. A novel compound, 2-deoxy-2-(N-carbamoylmethyl-[N'-2'-methyl-3'-hydroxypyrid-4'-one])-D-glucopyranose, designed to chelate Fe(3+) or Al(3+), has been synthesised utilising three naturally occurring products: maltol, glycine and glucosamine. The synthesis is a simple two step process. First, glycine is coupled to maltol by an aminolysis reaction, to yield the intermediate product 1-carboxymethyl -3-hdroxy-2-methylpyrid-4-one, which is joined with glucosamine using a dicyclohexylcarbodiimide promoted peptide coupling method to produce the desired end product, 2-deoxy-2-(N-carbamoylmethyl-[N'-2'-methyl-3'-hydroxypyrid-4'-one])-D-glucopyranose. The latter has been given the trivial name Feralex-G. NMR analysis permitted assignment of frequencies for all carbon and covalently bound hydrogen atoms and was consistent with the proposed structure of the compound. Electron spray Ionisation Mass Spectrometry (ESI-MS) yielded the expected molecular mass of 344. Proton displacement/pH titration analysis yielded three Feralex-G molecules bound to 1 Al(3+) or Fe(3+) over a measurable pH range of 3-10.5. A rapid TLC method to monitor progression of the synthetic procedures is also described.     

Amperometric sulfide detection using Coprinus cinereus peroxidase immobilized on screen printed electrode in an enzyme inhibition based biosensor

In the present work, an amperometric inhibition biosensor for the determination of sulfide has been fabricated by immobilizing Coprinus cinereus peroxidase (CIP) on the surface of screen printed electrode (SPE). Chitosan/acrylamide was applied for immobilization of peroxidase on the working electrode. The amperometric measurement was performed at an applied potential of -150 mV versus Ag/AgCl with a scan rate of 100 mV in the presence of hydroquinone as electron mediator and 0.1M phosphate buffer solution of pH 6.5. The variables influencing the performance of sensor including the amount of substrate, mediator concentration and electrolyte pH were optimized. The determination of sulfide can be achieved in a linear range of 1.09-16.3 μM with a detection limit of 0.3 μM. Developed sensor showed quicker response to sulfide compared to the previous developed sulfide biosensors. Common anions and cations in environmental water did not interfere with sulfide detection by the developed biosensor. Cyanide interference on the enzyme inhibition caused 43.25% error in the calibration assay which is less than the amounts reported by previous studies. Because of high sensitivity and the low-cost of SPE, this inhibition biosensor can be successfully used for analysis of environmental water samples.     

The effects of calcium and magnesium ions on the adenosine triphosphatase and inosine triphosphatase activities of myosin A

1. The effects of Ca(2+) and Mg(2+) on the enzymic activity of myosin were studied with myosin preparations treated by the ion-exchange resin Chelex-100. A reaction mixture containing 0.05m-potassium chloride was chosen in which the effects of univalent ions such as K(+), Na(+) and Cl(-) do not change significantly with small variations in their concentrations. 2. The relationship between the rate of hydrolysis of ATP or ITP and the concentration of Ca(2+) suggests that a relatively weak binding of Ca(2+) either to myosin or to the substrate nucleotide is responsible for the activation of the enzymic activity. According to the experiments with an ultrafiltration technique, the binding of Ca(2+) to myosin proceeds in at least two steps, the first occurring at one site on every 500000 atomic mass units of myosin with an apparent association constant, K(app.), 1.3x10(6)m(-1), and the second seeming to be so weak that its binding parameters cannot be determined by the method used. The first type of Ca(2+) binding is not observable with N-ethylmaleimide-modified myosin, yet this modified myosin shows activation by Ca(2+) of its adenosine triphosphatase and inosine triphosphatase. 3. The inhibition by Mg(2+) can be related to a binding reaction of Mg(2+) with myosin having K(app.) approximately 10(6)m(-1). Mg(2+) replaces the Ca(2+) bound tightly to myosin. The K(app.) for Mg(2+)-myosin binding calculated by assuming a competition between Ca(2+) and Mg(2+) for the same site is 2.1x10(5)-3.0x10(5)m(-1). When myosin is modified with a thiol reagent (p-mercuribenzoate) at a certain ratio to myosin, the inhibition by Mg(2+) becomes unobservable. 4. The behaviour of the hydrolytic activity of myosin on ATP or ITP in the presence of both Ca(2+) and Mg(2+) is consistent with the explanation that the inhibition by Mg(2+) is due to the tight binding of Mg(2+) to myosin, whereas the activation by Ca(2+) is caused either by a weak binding of Ca(2+) to myosin or by CaATP(2-) or by both.     

Improved experimental and computational methodology for determining the kinetic equation and the extant kinetic constants of Fe(II) oxidation by Acidithiobacillus ferrooxidans

The variety of kinetics expressions encountered in the literature and the unreasonably broad range of values reported for the kinetics constants of Acidithiobacillus ferrooxidans underscore the need for a unifying experimental procedure and for the development of a reliable kinetics equation. Following an extensive and critical review of reported experimental techniques, a method based on batch pH-controlled kinetics experiments lasting less than one doubling time was developed for the determination of extant kinetics constants. The Fe(II) concentration in the experiments was measured by a method insensitive to Fe(III) interference. Kinetics parameters were determined by nonlinear fitting of the integrated form of the Monod equation to yield a K(S) of 31 +/- 4 mg Fe(2+) liter(-1) (mean +/- standard deviation), a K(P) of 139 +/- 20 mg Fe(3+) liter(-1), and a mu(max) of 0.082 +/- 0.002 h(-1). The corresponding kinetics equation was as follows: dSdt=-0.0822.3.10(7)S.X31(1+P(0)+S(0)-S139)+S where S represents the Fe(II) concentration in mg liter(-1), P(0) represents the initial Fe(III) concentration in mg liter(-1), X represents the suspended bacterial cell concentration in cells ml(-1), and t represents time in hours. The measured data fit this equation exceptionally well, with an R(2) of >0.99. Fe(III) inhibition was found to be of a competitive nature. Contrary to previous reports, the results show that the concentration of Acidithiobacillus ferrooxidans cells has no affect on the kinetics constants. The kinetics equation can be considered applicable only to A. ferrooxidans cells grown under environmental conditions similar to those of the inoculum tested in the study. In contrast, the experimental and computational procedure is completely general and can be applied to A. ferrooxidans irrespective of the culture history.