Addition Of Thiosulfate Research Articles

Promotion of the microalgal photo‐biocatalytic asymmetric reduction of prochiral ketone by NADPH metabolic regulation

AbstractA metabolic regulation method was proposed to increase the microalgal intracellular nicotinamide adenine dinucleotide phosphate (NADPH) by lowering NADPH consumption in the Calvin cycle or accelerating NADPH regeneration in photosynthesis, thereby to promote the asymmetric reduction of prochiral ketone to chiral alcohol catalyzed by microalgae cell. The NADPH consumption was efficiently decreased by inhibiting glyceraldehyde 3‐phosphate dehydrogenase with inhibitors, such as iodoacetic acid and Angeli's Salt, which increased the microalgal intracellular NADPH by 80% and 96%, respectively. Correspondingly, the yields of the asymmetric reduction reaction were enhanced by 12.9% and 63.3%, respectively. On the other hand, NADPH regeneration was accelerated by promoting electron transfer and ferredoxin‐NADP+ reductase activity with addition of sodium cholate, sodium acetate and sodium thiosulfate; the intracellular NADPH was increased 73.1%, 60.2% and 70.4%, respectively. The yields of the asymmetric reduction reaction were correspondingly enhanced by 118.4%, 36.7% and 26.2%, respectively. This provides a practical technique to promote microalgae intracellular NADPH amount to enhance asymmetric reduction reaction by photo‐biocatalytic processes. © 2016 Curtin University of Technology and John Wiley & Sons, Ltd.

Phytoremediation of a multi contaminated soil: mercury and arsenic phytoextraction assisted by mobilizing agent and plant growth promoting bacteria

Purpose The possibility of using chemical and microbial additives to enhance the phytoextraction of mercury (Hg) and arsenic (As) from a multi-contaminated soil could be very effective, leading to a significant saving in terms of time and costs of the reclamation. The aim of this study was to evaluate the efficacy of the addition of (i) thiosulfate and (ii) metal-tolerant bacteria isolated from the polluted soil having plant growth promotion (PGP) potential to perform As and Hg phytoextraction by Brassica juncea and Lupinus albus.

Enrichment of Desulfitobacterium spp. from forest and grassland soil using the O-demethylation of phenyl methyl ethers as a growth-selective process.

The O-demethylation of phenyl methyl ethers under anaerobic conditions is a metabolic feature of acetogens and Desulfitobacterium spp. Desulfitobacteria as well as most acetogens are Gram-positive bacteria with a low GC content and belong to the phylum Firmicutes. The consumption of the phenyl methyl ether syringate was studied in enrichment cultures originating from five different topsoils. Desulfitobacterium spp. were detected in all topsoils via quantitative PCR. Desulfitobacteria could be enriched using the O-demethylation of syringate as a growth-selective process. The enrichment was significantly favoured by an external electron acceptor such as 3-chloro-4-hydroxyphenylacetate or thiosulfate. Upon cultivation in the presence of syringate and thiosulfate, which naturally occur in soil, a maximum number of 16S rRNA gene copies of Desulfitobacterium spp. was reached within the first three subcultivation steps and accounted for 3-10% of the total microbial community depending on the soil type. Afterwards, a loss of Desulfitobacterium gene copies was observed. Community analyses revealed that Proteobacteria, Acidobacteria, Actinobacteria and Bacteroidetes were the main phyla in the initial soil samples. Upon addition of syringate and thiosulfate as growth substrates, these phyla were rapidly outcompeted by Firmicutes, which were under-represented in soil. The main Firmicutes genera identified were Alkalibaculum, Clostridium, Sporobacterium, Sporomusa and Tissierella, which might be responsible for outcompeting the desulfitobacteria. Most of these organisms belong to the acetogens, which have previously been described to demethylate phenyl methyl ethers. The shift of the native community structure to almost exclusively Firmicutes supports the participation of members of this phylum in environmental demethylation processes.

Mechanism of Thiosulfate Oxidation in the SoxA Family of Cysteine-ligated Cytochromes

Thiosulfate dehydrogenase (TsdA) catalyzes the oxidation of two thiosulfate molecules to form tetrathionate and is predicted to use an unusual cysteine-ligated heme as the catalytic cofactor. We have determined the structure of Allochromatium vinosum TsdA to a resolution of 1.3 Å. This structure confirms the active site heme ligation, identifies a thiosulfate binding site within the active site cavity, and reveals an electron transfer route from the catalytic heme, through a second heme group to the external electron acceptor. We provide multiple lines of evidence that the catalytic reaction proceeds through the intermediate formation of a S-thiosulfonate derivative of the heme cysteine ligand: the cysteine is reactive and is accessible to electrophilic attack; cysteine S-thiosulfonate is formed by the addition of thiosulfate or following the reverse reaction with tetrathionate; the S-thiosulfonate modification is removed through catalysis; and alkylating the cysteine blocks activity. Active site amino acid residues required for catalysis were identified by mutagenesis and are inferred to also play a role in stabilizing the S-thiosulfonate intermediate. The enzyme SoxAX, which catalyzes the first step in the bacterial Sox thiosulfate oxidation pathway, is homologous to TsdA and can be inferred to use a related catalytic mechanism.

Effect of Thiosulfate on Pitting Corrosion of 316SS: II. Metastable Pitting and Transition to Stability

In the present part of study, the effect of thiosulfate ion on pitting corrosion of AISI 316 stainless steel in 0.1 M NaCl solution was investigated. In order to elucidate the effect of thiosulfate on pitting corrosion, the metastable pitting has been studied in more details. The results showed that the thiosulfate addition increased susceptibility to pit initiation and increased the dissolution kinetics of metastable growing pits in a fashion that facilitates the transition from metastability to stability. Consequently, the pitting potential of the stainless steel was decreased in the presence of thiosulfate ion.

Effect of Thiosulfate on Pitting Corrosion of 316SS: I. Critical Pitting Temperature and Pit Chemistry

In this research, the effect of thiosulfate ion on pitting corrosion behavior of 316 stainless steel was studied. Part I of this work deals with the influence of thiosulfate on the critical pitting temperature of 316 SS in 0.1 M chloride solution and studies the effect of this ion on the pit chemistry. Electrochemical experiments performed in absence and presence of 0.01 M thiosulfate ion revealed that thiosulfate deteriorates the CPT. Pencil electrode studies showed that thiosulfate addition decreases both saturation concentration of metal cations necessary to metal salt precipitate at the pit bottom (CS) (the value of diffusion controlled limiting current density (iLim) was decreased) and the ratio of C*/CS (where C* is the critical concentration of metal cations in pit solution essential for a pit to remain stable) while it increases the anodic dissolution rate in a simulated pit solution. Furthermore, scanning electron microscopy showed that the lacy cover formed over the pits mouth has coarser pore structure in the presence of thiosulfate ion.

Fast conformational exchange between the sulfur-free and persulfide-bound rhodanese domain of E. coli YgaP

Rhodanese domains are abundant structural modules that catalyze the transfer of a sulfur atom from thiolsulfates to cyanide via formation of a covalent persulfide intermediate that is bound to an essential conserved cysteine residue. In this study, the three-dimensional structure of the rhodanese domain of YgaP from Escherichia coli was determined using solution NMR. A typical rhodanese domain fold was observed, as expected from the high homology with the catalytic domain of other sulfur transferases. The initial sulfur-transfer step and formation of the rhodanese persulfide intermediate were monitored by addition of sodium thiosulfate using two-dimensional 1H–15N correlation spectroscopy. Discrete sharp signals were observed upon substrate addition, indicting fast exchange between sulfur-free and persulfide-intermediate forms. Residues exhibiting pronounced chemical shift changes were mapped to the structure, and included both substrate binding and surrounding residues.

Dynamics of metabolic activities and gene expression in the Roseobacter clade bacterium Phaeobacter sp. strain MED193 during growth with thiosulfate.

Metagenomic analyses of surface seawater reveal that genes for sulfur oxidation are widespread in bacterioplankton communities. However, little is known about the metabolic processes used to exploit the energy potentially gained from inorganic sulfur oxidation in oxic seawater. We therefore studied the sox gene system containing Roseobacter clade isolate Phaeobacter sp. strain MED193 in acetate minimal medium with and without thiosulfate. The addition of thiosulfate enhanced the bacterial growth yields up to 40% in this strain. Concomitantly, soxB and soxY gene expression increased about 8-fold with thiosulfate and remained 11-fold higher than that in controls through stationary phase. At stationary phase, thiosulfate stimulated protein synthesis and anaplerotic CO2 fixation rates up to 5- and 35-fold, respectively. Several genes involved in anaplerotic CO2 fixation (i.e., pyruvate carboxylase, propionyl coenzyme A [CoA], and crotonyl-CoA carboxylase) were highly expressed during active growth, coinciding with high CO2 fixation rates. The high expression of key genes in the ethylmalonyl-CoA pathway suggests that this is an important pathway for the utilization of two-carbon compounds in Phaeobacter sp. MED193. Overall, our findings imply that Roseobacter clade bacteria carrying sox genes can use their lithotrophic potential to gain additional energy from sulfur oxidation for both increasing their growth capacity and improving their long-term survival.

Effects of Thiosulfate on the Adsorption of Arsenate on Hematite With a View to Phytoextraction

A study was carried out to evaluate how the adsorption process of arsenate on hematite is influenced by the presence of thiosulfate ion. The competition in sorption between the two anions was examined in relation to potential use of thiosulfate as an additive for phytoextraction in arsenic contaminated soils. Results obtained from sorption isotherms can be described according to the Langmuir equation which allows, by comparing adsorption maxima, a quantitative evaluation of the effect of increasing concentration of thiosulfate on arsenate adsorption. Due to the competition between the two anions for available sites on hematite, arsenic sorption decreased at increasing thiosulfate concentration. The negligible effect of ionic strength on the sorption process, strongly suggests that thiosulfate can substitute arsenate ions in the inner sphere complexes on the surface of hematite. The interpretation of the As adsorption process on hematite in terms of distribution coefficient (K d) shows that thiosulfate ions competing for sorption on the same surface may significantly reduce the arsenate adsorption and enhance the release of arsenic in the liquid phase of soil. Results have a direct influence on the applicability of phytoextraction in arsenic contaminated soils since plants can uptake arsenic only from the liquid phase. The obtained results can explain the increase in As concentration in plants, reported in a recent paper in which the addition of thiosulfate was shown to promote As phytoavailability.

Testing the degradation effects of three reagents on various antineoplastic compounds.

Studies for decontamination of antineoplastic compounds have been conducted for decades. Nevertheless, recent studies indicate the contamination of work place in hospitals, and the exposure of workers. In this study, to develop an effective cleaning method for contaminated environments, the degradation efficacies of antineoplastic compounds by reagents were evaluated. The degradation efficacies of various combinations of three reagents (sodium hypochlorite, sodium thiosulfate, and sodium hydroxide) were evaluated with four antineoplastic compounds (cyclophosphamide, epirubicin, cisplatin, and carboplatin). The residues of antineoplastic compounds were measured with high-performance liquid chromatography. Of the three reagents, sodium hypochlorite was the most effective to all antineoplastic compounds used in this study. Although sodium hypochlorite degraded 86.6% of cyclophosphamide, it degraded other three antineoplastic compounds completely. The combination with sodium hypochlorite and sodium thiosulfate degraded only 3.3% of cyclophosphamide, since sodium thiosulfate inhibited the degradation ability of sodium hypochlorite. Similarly, the combinations used in all three reagents failed to degrade cyclophosphamide. Although three of four antineoplastic compounds were degraded successfully, any combinations of three reagents could not degrade cyclophosphamide completely. However, the addition of sodium thiosulfate which inhibits the corrosion of metal by sodium hypochlorite is essential for daily cleaning. Therefore, the evaluation of reaction time before the addition of sodium thiosulfate may be required. We will continue to investigate the reagents for degradation.

The Effect of Thiosulphate on Arsenic Bioavailability in a Multi Contaminated Soil. A Novel Contribution to Phytoextraction

In this study it was evaluated on a laboratory scale (microcosm), the possibility of using ammonium thiosulphate in assisted phytoextraction for the simultaneous removal of mercury and arsenic from multi- contaminated industrial soil. The species selected were Brassica juncea and Lupinus albus the addition of thiosulfate to the soil greatly promoted the uptake and translocation of both contaminants in the aerial parts of the plants. Mercury concentration in the aerial parts reached in B. juncea 120 mg/kg approximately 40 times the value of the control). The concentration of arsenic also increased significantly in the shoots of B. juncea (14.3 mg/kg), where the value in the control was negligible. Similar trends were obtained for L. albus The results confirmed the known positive effects of thiosulphate in increasing mercury bioavailability for plants, moreover they showed the ability of thiosulphate to mobilize arsenic and significantly to promote its uptake by plants. The increase of arsenic bioavailability, promoted by thiosulfate addition, could be attributed to the competition between arsenate and sulphate ions for the same active sites in the soil surfaces, with the release of arsenic in the soil solution. The use of thiosulfate appears to have great potential since it is a common fertilizer used to promote plant growth and is able to increase the uptake by plants of mercury and arsenic. The simultaneous increase of both contaminants uptake by plants, using a single additive, will provide new insights into the phytoextraction technology in terms of cost and time reduction.

Natural antifouling compounds produced by a novel fungus Aureobasidium pullulans HN isolated from marine biofilm

A fungus, Aureobasidium pullulans, was isolated from marine biofilm and identified. A bioassay-guided fractionation procedure was developed to isolate and purify antifouling compounds from A. pullulans HN. The procedure was: fermentation broth—aeration and addition of sodium thiosulfate–graduated pH and liquid–liquid extraction—SPE purification—GC–MS analysis. Firstly, the fermentation broth was tested for its toxicity. Then it was treated with aeration and addition of sodium thiosulfate, and its toxicity was almost not changed. Lastly, antifouling compounds were extracted at different pH, the extract had high toxicity at pH 2 but almost no toxicity at pH 10, which suggested the toxicants should be fatty acids. The EC50 of the extract against Skeletonema costatum was 90.9μgml−1, and its LC50 against Balanus amphitrete larvae was 22.2μgml−1. After purified by HLB SPE column, the EC50 of the extract against S. costatum was 49.4μgml−1. The myristic and palmitic acids were found as the main toxicants by GC–MS.

A mechanistic study on thiosulfate-enhanced passivity degradation of Alloy 800 in chloride solutions

This paper reports thiosulfate-enhanced effect of chloride ions on the passivity degradation of Alloy 800 and discusses how thiosulfate influences the semiconductor properties as well as breakdown of the passive films. Experimental results reveal that in concentrations of 10−6 to 0.075mol/L, thiosulfate lowers the breakdown potential of Alloy 800 by 100–400mV in solutions containing 0.6mol/L chloride ions. This degradation is further verified by polarization resistance and noise resistance obtained from electrochemical impedance spectroscopy (EIS) and micro-nanoscale electrochemical noise (EN) data, respectively. Mott–Schottky analysis is employed to explore the corrosion mechanism and the results show that the passive film exhibits as p-type semiconductivity in a thiosulfate-only solution and n-type semiconductivity in a chloride-only solution, and the addition of thiosulfate into chloride solutions increases the concentration of oxygen vacancies in the passive film at the potential close to the pitting potential. Thiosulfate cannot be easily reduced on well-passivated surfaces in chloride-free solutions but can be readily reduced to sulfide in chloride solutions at active sites when the passive film breaks down – the reduced sulfur retards the healing process of repassivation, leading to an increase in oxygen vacancies.

P63: The antidotal action of nitrites toward cyanide intoxication: An example of medicine disguised as quackery

Sodium nitrite ameliorates sub-lethal cyanide toxicity in mice when given from ∼1 h before until 20 min after the toxic dose as demonstrated by the recovery of righting ability. In most experiments nitrite was given intraperitoneally, but may also be administered as an inhaled aqueous vapor-contrary to recommended EMR protocols, it is not necessary to infuse the antidote intravenously and the co-administration of adjuvants (specifically thiosulfate) is unnecessary. Intraperitoneally administered nitrite rapidly produces NO in the bloodstream as judged by the dose-dependent appearance of EPR signals attributable to nitrosylhemoglobin and methemoglobin. However, the commonly held belief that the antidotal mechanism of nitrites involves the scavenging of cyanide anion by methemoglobin is firmly contraindicated by recent evidence. Intriguingly, the FDA-approved and clinically tested cyanide scavenger cobalamin has, in fact, rather indifferent binding affinity for cyanide anion. It follows that the clinical usefulness of cobalamin may be dependent on endogenous nitric oxide displacing the cyanide from the active site of cytochrome c oxidase to facilitate the cyanide-scavenging action of cobalamin. Antagonism of cyanide inhibition of cytochrome c oxidase by NO appears to be the crucial antidotal activity rather than any methemoglobin-forming action of nitrite. Concomitant addition of sodium thiosulfate to nitrite-treated blood results in the production of sulfidomethemoblobin as detected by EPR spectroscopy. Sulfide is a product of thiosulfate hydrolysis and, like cyanide, is known to be a potent inhibitor of cytochrome c oxidase; the effects of the two inhibitors being essentially additive under standard assay conditions, rather than dominated by either one. The findings afford a plausible explanation for an observed detrimental effect in mice associated with the use of the standard nitrite-thiosulfate combination therapy at sub-lethal levels of cyanide intoxication. Isoamyl nitrite, given intraperitoneally in equimolar amounts to sodium nitrite, is comparatively less effective at restoring the righting recovery of cyanide-challenged mice. The results of investigations with the hydrolysis products of isoamyl nitrite, isoamyl alcohol and nitrite anion, suggest that the toxicity of isoamyl alcohol renders the organic nitrite a less desirable antidotal agent than sodium nitrite. Disclosure Supported by the CounterACT Program, National Institutes of Health Office of the Director (NIH OD), and the National Institute of Neurological Disorders and Stroke (NINDS), Grant No. NS063732 to J.P and L.L.P and Bruce R. Pitt.

Speciation of Silver Nanoparticles and Silver(I) by Reversed-Phase Liquid Chromatography Coupled to ICPMS

The tremendous increase in the use of engineered nanoparticles in daily life has raised concerns about their impact on the environment and in biological systems. Among them, silver-containing material is of high industrial interest and of manifold use in consumer products, mainly because of their antimicrobial activity. Therefore, analytical methods are urgently needed for the reliable determination of Ag nanoparticles and their corresponding Ag(I) species. In this study, we present the development of coupling reversed-phase high-performance liquid chromatography (HPLC) to inductively coupled plasma-mass spectrometry (ICPMS) for the speciation of engineered Ag-containing nanoparticles and Ag(I) species. The method has been designed for the separation/detection of all investigated silver species in a single chromatographic run. For this purpose, the addition of thiosulfate to the mobile phase has been used to elute Ag(I) species from the column without degradation of the other species. The analytical figures of merit show repeatable results for the recoveries (>80%) of both, the Ag nanoparticles and Ag(I) species. The obtained detection limits are in the medium ng·L(-1) range and therefore allow the trace analysis of the sought analytes in real samples. However, the matrix (e.g., fetal bovine serum) showed an impact on the retention behavior of the Ag nanoparticles, so that for size determinations the use of gold nanoparticles as internal size standard is suggested. Finally, the analysis of textile products exemplarily demonstrates the applicability to the analysis of real samples. Besides silver-containing nanoparticles, Ag(I) species can be identified as one of the major species in the extraction solution from sports socks. However, extraction conditions will be the subject of further investigations in the future in order to obtain reliable quantitative data.

Acute, Sublethal Cyanide Poisoning in Mice Is Ameliorated by Nitrite Alone: Complications Arising from Concomitant Administration of Nitrite and Thiosulfate as an Antidotal Combination

Sodium nitrite alone is shown to ameliorate sublethal cyanide toxicity in mice when given from ∼1 h before until 20 min after the toxic dose as demonstrated by the recovery of righting ability. An optimum dose (12 mg/kg) was determined to significantly relieve cyanide toxicity (5.0 mg/kg) when administered to mice intraperitoneally. Nitrite so administered was shown to rapidly produce NO in the bloodsteam as judged by the dose-dependent appearance of EPR signals attributable to nitrosylhemoglobin and methemoglobin. It is argued that antagonism of cyanide inhibition of cytochrome c oxidase by NO is the crucial antidotal activity rather than the methemoglobin-forming action of nitrite. Concomitant addition of sodium thiosulfate to nitrite-treated blood resulted in the detection of sulfidomethemoblobin by EPR spectroscopy. Sulfide is a product of thiosulfate hydrolysis and, like cyanide, is known to be a potent inhibitor of cytochrome c oxidase, the effects of the two inhibitors being essentially additive under standard assay conditions rather than dominated by either one. The findings afford a plausible explanation for an observed detrimental effect in mice associated with the use of the standard nitrite-thiosulfate combination therapy at sublethal levels of cyanide intoxication.

Adventitious shoot regeneration from hypocotyl slices of mature apricot ( Prunus armeniaca L.) seeds: A feasible alternative for apricot genetic engineering

Adventitious shoot regeneration from hypocotyl slices of mature apricot seeds has been achieved with regeneration percentages of 31.7%, 44.4%, and 46.9% for the cultivars ‘Canino’, ‘Dorada’, and ‘Moniqui’, respectively. Regeneration was significantly affected by the parental origin of the explants ( P < 0.05) but not by thidiazuron or 3-indolebutyric acid for any of the three cultivars, at the levels tested. None of the other factors studied (basal medium, 2,4 dichlorophenoxy-acetic acid pulses, dark incubation period, or addition of silver thiosulfate) affected significantly shoot regeneration percentage from ‘Canino’ hypocotyl sections. The effect of paromomycin on regeneration was genotype-dependent and different dose–response curves were obtained for each cultivar. While 40 μM paromomycin completely inhibited regeneration from ‘Canino’ sections, some buds were obtained from ‘Dorada’ and ‘Moniqui’ explants. The two aminoglycoside antibiotics tested, kanamycin and paromomycin, showed differing toxicity on ‘Canino’. A lower concentration of kanamycin (20 μM) than of paromomycin inhibited totally adventitious regeneration from ‘Canino’ explants. Agrobacterium-mediated transformation experiments, with the non-oncogenic strain AGL1 harboring the binary plasmid p35SGUSINT, were performed and GUS assays were carried out after four weeks to determinate stable transformation events. The utilization of paromomycin (10 μM) as the selective agent increased significantly both the number of explants that presented at least one transformation event ( P < 0.05) and the number of large area or calli expressing the gus gene ( P < 0.001), compared with the addition of kanamycin (10 μM). Moreover, when 10 μM paromomycin was added to the medium some massively transformed explants were observed and a chimerical bud was regenerated.

Growth of the Cyanobacterium Synechococcus leopoliensis CCAP1405/1 on Agar Media in the Presence of Heterotrophic Bacteria

The cyanobacterium Synechococcus leopoliensis CCAP1405/1 does not grow on common solid media made of agar, agarose HT, noble agar, gelrite and gelatin, although it grows in liquid media with the same components. The inoculation of S. leopoliensis CCAP1405/1 at a high initial cell density allowed it to grow on the agar media, and co-inoculation with one of the heterotrophic bacterial strains belonging to a wide range of phylogeny, showed the same effects even at a low initial cell density of S. leopoliensis CCAP1405/1. The addition of thiosulfate and high concentrations of vitamin B(12), biotin and thiamine also supported growth on solid media, but catalase had no effect. On inorganic solid media, the autotrophic cyanobacterial strain supported the growth of heterotrophic bacteria, suggesting mutual interaction.

A rhodanese-like protein is highly overrepresented in the mutant S. clavuligerus oppA2::aph: effect on holomycin and other secondary metabolites production.

SummaryA protein highly overrepresented in the proteome of Streptomyces clavuligerus oppA2::aph was characterized by MS/MS as a rhodanese‐like enzyme. The rhlA gene, encoding this protein, was deleted from strains S. clavuligerus ATCC 27064 and S. clavuligerus oppA2::aph to characterized the RhlA enzyme activity, growth on different sulfur sources and antibiotic production by the mutants. Whereas total thiosulfate sulfurtransferase activity in cell extracts was not affected by the rhlA deletion, growth, cephamycin C and clavulanic acid production were impaired in the rhlA mutants. Holomycin production was drastically reduced (66–90%) in the rhlA mutants even when using S. clavuligerusΔrhlA pregrown cells, suggesting that this enzyme might be involved in the formation of the cysteine precursor for this sulfur‐containing antibiotic. While growth on thiosulfate as the sole sulfur source was particularly low the volumetric and specific antibiotic production of the three antibiotics increased in all the strains in the presence of thiosulfate. This stimulatory effect of thiosulfate on antibiotic production was confirmed by addition of thiosulfate to pre‐grown cells and appears to be a general effect of thiosulfate on oxidative stress as was also evident in the production of staurosporin by S. clavuligerus.

Determination of Copper and Zinc in Brass: Two Basic Methods

In this experiment, the concentrations of copper and zinc in brass are obtained by two methods. This experiment does not require advanced instrumentation, uses inexpensive chemicals, and can be easily carried out during a 3-h upper-level undergraduate laboratory. Pedagogically, the basic concepts of analytical chemistry in solutions, such as pH, solubility, complexation, and redox reactions, are illustrated. In addition, this experiment can be used to introduce the science of alloys. Oxidation of brass pieces by concentrated nitric acid yields a solution of Cu2+ and Zn2+ cations. The first method consists in an acid−base titration of Cu2+ and Zn2+ cations by sodium hydroxide. The successive precipitations of the corresponding hydroxide complexes are observed in a pH titration curve that allows the calculation of the concentrations of Cu2+ and Zn2+ and thus the brass composition. The second method is based on complexometric titrations with and without the competition between complexing agents, steps 1 and 2, respectively. In step 1, the copper and zinc together are titrated by EDTA. In step 2, the addition of sodium thiosulfate masks the Cu2+ and the titration by EDTA leads to the determination of Zn2+ concentration. Cu2+ concentration and brass composition are readily obtained.