A selective and sensitive fluorescent sensor for cysteine detection based on bi‐8‐carboxamidoquinoline derivative and Cu2+ complex

In this paper, a novel fluorescent sensor 1 for selective and sensitive detection of cysteine was developed based on a complex between bi‐8‐carboxamidoquinoline derivative ligand ( L ) and Cu²⁺. The interaction of Cu²⁺ with the ligand causes a dramatic fluorescence quenching most likely due to its high affinity towards Cu²⁺ and a ligand–metal charge transfer (LMCT) process. The in situ generated L–Cu ² complex was utilized as a chemosensing ensemble for cysteine. In the presence of cysteine, the fluorophore, L , was released from L–Cu ² complex because of the strong affinity of cysteine to Cu²⁺ via the Cu–S bond, leading to the fluorescence recovery of the ligand. The proposed displacement mechanism was confirmed by the results of mass spectrometry (MS) study. Under optimized conditions, the recovered fluorescence intensity is linear with cysteine concentrations in the range 1 × 10^?6 mol/l to 8 × 10^?6 mol/l. The detection limit for cysteine is 1.92 × 10^?7 mol/l. Furthermore, the established method showed a highly sensitive and selective response to cysteine among the 20 fundamental α‐amino acids used as the building blocks of proteins, after Ni²⁺ was used as a masking agent to eliminate the interference of His. The proposed sensor is applicable in monitoring cysteine in practical samples with good recovery rate.     

Increasing copper alters cellular elemental composition (Mo and P) of marine diatom

The elemental composition (surface adsorbed and internalized fraction of Cu, Mo and P) in marine phytoplankton was first examined in cultures of the diatom Phaeodactylum tricornutum which were exposed to various levels of Cu concentrations ranging from 0.25 to 16 μmol/L with equivalent free [Cu²⁺] concentrations of 0.4–26 nmol/L. We observed an acceleration of algal growth rates (20–40%) with increasing ambient Cu levels, as well as slightly increased levels of internalized Cu in cells (2–13 × 10^?18 mol/cell) although cellular Cu mostly accumulated onto the cell surface (>50% of the total: intracellular + surface adsorbed). In particular, we documented for the first time that the elemental composition (Mo and P) in algal cells varies dynamically in response to increased Cu levels: (1) Cellular P, predominantly in the intracellular compartment (>95%), shows with a net consumption as indicated by a gradual decrease with increasing [Cu²⁺] (120→50 × 10^?15 mol P/cell) probably due to the fact that P, a backbone bioelement, is largely required in forming biological compartments such as cell membranes; and (2) cellular Mo, predominantly encountered in the intracellular compartment, showed up to tenfold increase in concentration in the cultures exposed to Cu, with a peak accumulation of 1.1 × 10^?18 mol Mo/cell occurring in the culture exposed to [Cu²⁺] at 3.7 nmol/L. Such a net cellular Mo accumulation suggests that Mo might be specifically required in biological processes, probably playing a counteracting role against Cu.     

Re-evaluating the effects of organic ligands on copper toxicity to barley root elongation in culture solution

Abstract

In the presence of weak ligands, both free ion activity and organic complexes of Cu should b considered when predicting Cu toxicity in aquatic and soil-plant systems. However, there is littl information about the quantitative contribution of Cu that is organically complexed to Cu toxicity. In thi study, a bioassay using barley root elongation in culture solution was used to investigate the effects o organic ligands with different conditional stability constants on Cu toxicity and the quantitativ contribution of the organically complexed Cu to the Cu toxicity. The results indicated that a significan decrease (p<0.05) in Cu toxicity, assessed by barley root elongation, was observed in response to th addition of organic ligands. The decrease differed, to some extent, with different organic ligands o disodium ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), oxalate and malate at low and constant free Cu²⁺ activity. Addition of EDTA or NTA resulted in strong reduction of Cu toxicity while modest reduction of Cu toxicity was observed for the addition of malate as the relatively wea ligand. Furthermore, the results of the present study revealed that the CuNTA^? and CuEDTA^2? complexes were not toxic, while the Cu–malate complexes were mildly toxic to barley root elongation More importantly, it was found that the toxicity of Cu–malate complexes were nearly 0.5-fold less than that of free Cu²⁺ ions.     

The behaviors of metal ions in the CdTe quantum dots–H2O2 chemiluminescence reaction and its sensing application

The behaviors of 15 kinds of metal ions in the thiol‐capped CdTe quantum dots (QDs)–H₂O₂ chemiluminescence (CL) reaction were investigated in detail. The results showed that Ag⁺, Cu²⁺ and Hg²⁺ could inhibit CdTe QDs and H₂O₂ CL reaction. A novel CL method for the selective determination of Ag⁺, Cu²⁺ and Hg²⁺ was developed, based on their inhibition of the reaction of CdTe QDs and H₂O₂. Under the optimal conditions, good linear relationships were realized between the CL intensity and the logarithm of concentrations of Ag⁺, Cu²⁺ and Hg²⁺. The linear ranges were from 2.0 × 10^?6 to 5.0 × 10^?8 mol L^?1 for Ag⁺, from 5.0 × 10^?6 to 7.0 × 10^?8 mol L^?1 for Cu²⁺ and from 2.0 × 10^?5 to 1.0 × 10^?7 mol L^?1 for Hg²⁺, respectively. The limits of detection (S/N = 3) were 3.0 × 10^?8, 4.0 × 10^?8 and 6.7 × 10^?8 mol L^?1 for Ag⁺, Cu²⁺ and Hg²⁺, respectively. A possible mechanism for the inhibition of CdTe QDs and H₂O₂ CL reaction was also discussed. Copyright © 2009 John Wiley & Sons, Ltd.     

Response surface optimized peroxyoxalate chemiluminescence of octahydro‐Schiff base derivative as new luminophor and study of the quenching effect of some cations,amino acids and cholesterol

We report the first detailed study of the characteristics of an octahydro‐Schiff base derivative as a new luminophor in the peroxyoxalate chemiluminescence (POCL) system. The effect of reagents on this new POCL system was investigated. In addition, the response surface methodology was used for evaluating the relative significance of variables in this POCL system, establishing models and determining optimal conditions. The quenching effect of some cations and compounds such as Cu²⁺, Fe³⁺, Hg²⁺, imidazole, histidine and cholesterol on an optimized POCL reaction were studied. The dynamic ranges were up to approximaterly 100 and 175 × 10^?6 M for Cu²⁺ and cholesterol respectively. The detection limits were 3.3 × 10^?6 m and 2.58 × 10^?6 m for Cu²⁺ and histidine, respectively. In all cases the relative standard deviations were 4–5% (n = 4). Copyright © 2014 John Wiley & Sons, Ltd.     

A new benzimidazole‐based selective and sensitive ‘on–off’ fluorescence chemosensor for Cu2+ ions and application in cellular bioimaging

Two new twinborn benzimidazole derivates ( L and A ), which bonded pyridine via the ester space on the opposite and adjacent positions of the benzene ring of benzimidazole respectively, were designed and synthesized. Compound L displayed fluorescence quenching response only towards copper(II) ions (Cu²⁺) in acetonitrile solution with high selectivity and sensitivity. However, compound A presented ‘on–off’ fluorescence response towards a wide range of metal ions to different degrees and did not have selectivity. Furthermore, compound L formed a 1:1 complex with Cu²⁺ and the binding constant between sensor L and Cu²⁺ was high at 6.02 × 10⁴ M^?1. Job's plot, mass spectra, IR spectra, ¹H‐NMR titration and density functional theory (DFT) calculations demonstrated the formation of a 1:1 complex between L and Cu²⁺. Chemosensor L displayed a low limit of detection (3.05 × 10^?6 M) and fast response time (15 s) to Cu²⁺. The Stern–Volmer analysis illustrated that the fluorescence quenching agreed with the static quenching mode. In addition, the obvious difference of L within HepG2 cells in the presence and absence of Cu²⁺ indicated L had the recognition capability for Cu²⁺ in living cells.     

Effect of linear alkylbenzene sulfonate on Cu2+ removal by Spirulina platensis strain (FACHB‐834)

The removal efficiency of Cu²⁺ by Spirulina platensis (strain FACHB‐834), in viable and heat‐inactivated forms, was investigated in the presence and absence of linear alkylbenzene sulfonate (LAS). When the initial Cu²⁺ concentration was in the range of 0.5–1.5 mg · L^?1, a slight increase in growth rate of FACHB‐834 was observed. In contrast, when Cu²⁺ or LAS concentrations were at or higher than 2.0 or 6.0 mg · L^?1, respectively, the growth of FACHB‐834 was inhibited and displayed yellowing and fragmentation of filaments. The presence of LAS improved Cu²⁺ removal by ~20%, and accelerated attainment of Cu²⁺ retention equilibrium. For the 2‐ mg · L^?1 Cu²⁺ treatments, retention equilibrium occurred within 2 d and showed maximum Cu²⁺ removal of 1.83 mg · L^?1. In the presence of LAS, the ratio of extracellular bound Cu²⁺ to intracellular Cu²⁺ taken up by the cells was lower (1.05–2.26) than corresponding ratios (2.46–7.85) in the absence of LAS. The percentages of extracellular bound Cu²⁺ to total Cu²⁺ removal (both bound and taken up by cells) in the presence of LAS ranged from 51.2% to 69.3%, which was lower than their corresponding percentages (71.1%–88.7%) in the absence of LAS. LAS promoted biologically active transport of the extracellular bound form of Cu²⁺ into the cell. In contrast, the addition of LAS did not increase the maximum removal efficiency of Cu²⁺ (61.4% ± 5.6%) by heat‐inactivated cells compared to that of living cells (59.6% ± 6.0%). These results provide a theoretical foundation for designing bioremediation strategies using FACHB‐834 for use in surface waters contaminated by both heavy metals and LAS.     

The potentiation of industrial biocide activity with Cu2+. I. Synergistic effect of Cu2+ with formaldehyde

Earlier studies suggested that copper enhances the antimicrobial activity of some formaldehyde (FA)-condensate biocides in metalworking fluids as well as FA in laboratory media. The possible synergistic interaction between FA and Cu²⁺ in combination were tested against Pseudomonas aeruginosa in trypic soy broth, mineral salt base-glucose medium, and 0·9% NaCl solution. In all cases, Cu²⁺ enhanced the FA bactericidal activity. A sequential treatment of bacterial cultures was employed to study the increased effectiveness of the Cu²⁺ and FA combination. The cells were exposed to FA or Cu²⁺ and subsequently exposed to the alternate compound with centrifugation and washing between exposures. Results varied depending on the medium. Synergistic activity of FA and Cu²⁺ was established based on the interpretation of the results.     

Identifying the species of copper that are toxic to plant roots in alkaline nutrient solutions

Background and aims

The pH of the growth medium influences Cu speciation in solution, the negativity of plasma membrane (PM) surface potential, and hence the rhizotoxicity of Cu.

Methods

Solution culture experiments were conducted with wheat (Triticum aestivum L.) seedlings to examine the toxicity of various Cu species at pH values ranging from 4.50 to 8.25. The toxic species of Cu was identified, giving particular consideration to the electrical properties at the plant cell membrane and ion activities at the PM surface.

Results

The solution culture studies showed that at pH?<?6.60 (i.e., free Cu²⁺ >95 % of total Cu), the addition of cations (Ca²⁺ or H⁺) decreased the toxic effects of Cu by decreasing the negativity of the PM surface potential (and hence decreasing the activity of Cu²⁺ at the PM surface). For solutions with pH values from 7.50 to 8.25 (CuCO ⁰₃ >50 % of total Cu), an increase in pH significantly enhanced the toxicity of Cu, whilst the addition of Ca had negligible influence on toxicity.

Conclusions

Root growth in solution cultures was influenced primarily by the surface activities of free Cu²⁺ and CuCO ⁰₃ . Across all experiments, the data indicate that it was CuCO ⁰₃ , rather than CuOH⁺, that contributed Cu toxicity over pH?>?7.00. Although our data do not explore the mechanism of toxicity, we propose that CuCO ⁰₃ has an important role in Cu rhizotoxicity in alkaline growth media.

     

Metal cation toxicity in the alga Gracilaria domingensis as evaluated by the daily growth rates in synthetic seawater

Macroalgae of the genus Gracilaria have considerable economic importance as raw material for agar production and belong to an important group of organisms that are tolerant of high concentrations of metal. The median inhibitory concentration (IC₅₀) values obtained by measuring the ratio of fresh mass variation (i.e., daily growth rates) of the red macroalga Gracilaria domingensis during a 48-h aquatic toxicity assay are reported here. The alga was exposed to 14 different metal cations as well as the molybdate anion in synthetic seawater. The actual concentrations of these ionic species (at IC₅₀ values) and the proportion of free ions (aqueous complexes) were determined by inductively coupled plasma atomic emission spectroscopy and the Environmental Protection Agency-recommended software, MINTEQA2, respectively. Based on the free IC₅₀ values (IC₅₀ ^F), the ions were ranked in terms of toxicity: Cd²⁺???Cu²⁺???Pb²⁺???Zn²⁺???Ni²⁺?>?Co²⁺?>?La³⁺???Mn²⁺?>?Ca²⁺?~?Li⁺???MoO₄ ^2????Sr²⁺?>?Mg²⁺???K⁺?>?Na⁺. As a member of the first trophic level in the marine food chain, G. domingensis is an appropriate target organism both for the development of toxicological assays and as a bioindicator of marine degradation.     

A colorimetric and fluorescent chemosensor for selective detection of Cu2+ based on a new diarylethene with a benzophenone hydrazone unit

A new photochromic diarylethene based on benzophenone hydrazone has been synthesized. Its photochromic and fluorescent properties changed upon alternating irradiation with UV /Vis light and adding Cu²⁺/EDTA in methanol, which showed that the diarylethene could be served as a colorimetric and fluorescent chemosensor for selective detection of Cu²⁺ based on internal charge transfer processes. The colorimetric and turn‐off fluorescent selective detection of Cu²⁺ was attributed to the 2:1 complex of the diarylethene and Cu²⁺. The binding constant (Ka ) was 1.53 × 10⁴ L mol^?1 and the limit of detection of the diarylethene for Cu²⁺ was calculated to be 1.45 × 10^?6 mol L^?1. In addition, the metal‐responsive photochromic behavior of diarylethene was applied successfully to the construction of a molecular logic circuit.     

Binding of divalent copper ions to aspartic acid residue 52 in hen egg-white lysozyme

Divalent copper was found to inhibit non-competitively the lysis of Micrococcus lysodeikticus cells by hen egg-white lysozyme, with an inhibition constant K_a= 3.8 × 10²m^?1. The association constants of Cu²⁺ for lysozyme and for a derivative of lysozyme in which tryptophan residue 108 was selectively modified, were measured spectrofluorimetrieally and found to be 1.8 × 10²m^?1 and 1.0 × 10³m^?1, respectively. The electron spin resonance spectrum of Cu²⁺ was not affected by the addition of lysozyme, whereas many new lines appeared on addition of the modified protein. This was interpreted as evidence for the binding of Cu²⁺ in the neighbourhood of tryptophan 108. To unequivocally establish the site of ligation of Cu²⁺, crystals of lysozyme soaked in Cu²⁺ were examined by X-ray crystallography and the results compared to those obtained from crystals of native lysozyme. Cu²⁺ was found to be located 2 to 3 Å from the carboxyl side-chain of aspartic acid 52, 5 Å from the carboxyl of glutamic acid 35 and about 7 Å from tryptophan 108.The addition of a saccharide inhibitor to lysozyme was found to increase the association constant of Cu²⁺ for lysozyme from a value of 1.8 × 10²m^?1 to 6.0 × 10²m^?1. This finding was interpreted as indicative of a change in conformation around tryptophan 108 and glutamic acid 35 induced by the interaction of saccharides with the enzyme, which affects the metal binding properties of aspartic acid 52.     

Zinc and salinity effects on membrane transport in Chara connivens

Pressure-probe measurements showed that the pressure relaxation of internodal cells of the freshwater alga Chara connivens slowed considerably when 1–5 mol m^?3 Zn²⁺, or more especially Zn²⁺ and 75 mol m^?3 NaCl, were present in the medium for periods of 1 h or longer. These results indicate that the water permeability of the Chara membrane is decreased by Zn²⁺, and that this effect is enhanced by 75 mol m^?3 NaCl. Specific values taken after 375 min exposure were: 5 mol m^?3 Zn²⁺ and 75 mol m^?3 NaCl caused the half-time for bulk water movement to increase from 7·8±2·3 to 79·5±5·4s, corresponding to a decrease in the hydraulic conductivity (Lp) from (13·0±3·3) × 10^?7 m s^?1 mPa^?1 to (1·25±0·23) × 10^?7 m s^?1 MPa^?1 (mean±S.D., n= 10). These changes are not seen in the presence of NaCl alone, and to a reduced extent in the presence of 5 mol m^?3Zn²⁺ alone (after 375 min, Lp was (2·4±0·1) × 10^?7 m s^?1 MPa^?1, mean±S.D., n = 6). Ca²⁺ cannot substitute for Zn²⁺, but seems to competitively inhibit Zn²⁺. There was another, kinetically distinct effect of Zn²⁺: the ingress of Na⁺ within 15 min of exposure to 75 mol m^?3 NaCl is halved by the presence of 1–5 mol m^?3 Zn²⁺, although internal osmolality is little changed by Zn²⁺. In spite of this, Zn²⁺ does not exert the long-term protection against NaCl that has been reported for Ca²⁺. Depending on the concentration of Zn²⁺ and the duration of the exposure, the effects on water permeability were fully or partly reversible within 24–48 h. The mechanism of these changes is difficult to identify. One possibility is a zinc-induced restriction of trans-membrane channels to give single-file channels which can be blocked by salt.     

Enhanced electrochemiluminescence from reduced graphene oxide–CdTe quantum dots for highly selective determination of copper ion

An electrochemiluminescence (ECL) sensor based on reduced graphene oxide–CdTe quantum dots (RGO–CdTe QDs) composites for detecting copper ion (Cu²⁺) was proposed. The ECL behaviours of the RGO–CdTe QD modified electrode were investigated with H₂O₂ as the co‐reactant. Quantitative detection of Cu²⁺ was realized as Cu²⁺ could effectively quench the ECL signal of the RGO–CdTe QDs. A wide linear range of 1.00 × 10^?14 to 1.00 × 10^?4 M (R = 0.9953) was obtained under optimized conditions, and a detection limit (S/N = 3) was achieved of as low as 3.33 × 10^?15 M. The proposed sensor also exhibited good stability and selectivity for the detection of copper ions. Finally, the analytical application of the proposed sensor was also evaluated using river water.     

A colorimetric probe for detection of Cu2+ by the naked eye and application in test paper

A novel colorimetric probe RP1 was synthesized using rhodamine derivatives and heterocyclic compounds for the purpose of detecting Cu²⁺. RP1 showed good selectivity, high sensitivity and affinity toward Cu²⁺ over other competing ions in CH₃OH–H₂O (1/1, v/v) solution. Absorbance intensity showed a good linear fit between probe R1 and Cu²⁺ over the concentration range 1–8 μM and the association constant was also calculated to be 1.145 × 10⁵ M^?1. The sensing mechanism was deduced using Job's plot, Fourier transform infrared spectroscopy, and density functional theory studies. In addition, the colorimetric experiment indicated that probe RP1 could be made into test paper to detect Cu²⁺ with a colour change from colourless to pink.     

Copper complexation by natural organic matter in contaminated and uncontaminated ground water

Abstract

Ground-water samples were collected from an uncontaminated and a contaminated site. Copper complexation was characterized by ion- selective electrode (ISE), fluorescence quenching (FQ), and cathodic stripping voltammetric (CSV) titrations. All of the samples were titrated at their natural pH values and some of the samples were also titrated at other pH values. For a total Cu concentration of 10^?6 M, the free Cu²⁺ concentrations in the samples from the uncontaminated site were all less than 10^?7 M, while free Cu²⁺ in the samples from the contaminated site were all less than 10^?8 M. For a particular sample and total Cu concentration, the free Cu²⁺ concentration decreased as the pH increased. Relative to ISE, FQ underestimated and CSV overestimated the degree of Cu²⁺ binding. The Cu²⁺ -complexing properties of the ground waters are similar to many published results for the same pH and for ligand concentrations normalized to T.O.C. Chemical equilibrium computations indicate that organic complexes would dominate Cu speciation in the uncontaminated ground waters for 10^?7 to 10^?5 M total Cu. In the contaminated ground waters, sulfide complexes would be the predominant Cu species for total Cu less than the total S(?11) concentration. Organic complexes would dominate Cu speciation for total Cu greater than total S(?11).     

Effects of Combined Abiotic Stresses on Growth,Trace Element Accumulation,and Phytohormone Regulation in Two Halophytic Species

Trace element contamination of lands is a serious environmental problem that limits yield and threatens human health. To study the combined effect of high salinity and toxic levels of trace elements on halophytes, the performance of two marsh species, Atriplex halimus and Suaeda fruticosa, grown for 1 month with an irrigation solution supplemented with 200 mM NaCl and 400 μM Cd²⁺ or 400 μM Cu²⁺ was evaluated. The effect of the combined stress conditions on hormone signaling was also assessed. Biomass production and chlorophyll content decreased under Cd²⁺ stress in both species, whereas Cu²⁺ had a lower impact on plant performance. The different plant sensibilities to the two trace elements assayed indicate that each metal has a different effect on plants. Furthermore, the deleterious effect of metal toxicity was alleviated when NaCl was added to the irrigation solution, demonstrating that NaCl improves plant performance and tolerance of halophytic species to cope with trace element intoxication. Results show that both species accumulated important quantities of Cd²⁺ and Cu²⁺ in roots (Cd²⁺: 2,690–3,130 μg g^?1 DW and Cu²⁺: 2,070–2,770 μg g^?1 DW); this finding allows us to classify these species among the hyperaccumulator plants. Cd²⁺ and Cu²⁺ differently affected endogenous phytohormone contents in both species. Data suggest an essential involvement of roots on the regulation of tolerance to trace elements. Therefore, indole-3-acetic acid levels increased in roots of both species irrigated with high levels of Cd²⁺, which suggests that the auxin may stimulate root promotion and growth under these stress conditions. Other compounds, classically considered as “stress hormones” showed very different patterns of accumulation. Whereas, salicylic acid (SA) levels in roots and leaves increased in response to Cd²⁺, root contents of jasmonic acid (JA), and abscisic acid (ABA) decreased. In leaves, the rambling pattern of accumulation observed for JA and ABA suggested the lack of a specific role in regulation against trace element toxicity. Together, data suggest that SA could act as a specific signal that detects trace element toxicity, whereas JA and ABA promote general responses against abiotic stress.     

Fluorescent probe for detection of Cu2+ using core‐shell CdTe/ZnS quantum dots

Core‐shell CdTe/ZnS quantum dots capped with 3‐mercaptopropionic acid (MPA) were successfully synthesized in aqueous medium by hydrothermal synthesis. These quantum dots have advantages compared to traditional quantum dots with limited biological applications, high toxicity and tendency to aggregate. The concentration of Cu²⁺ has a significant impact on the fluorescence intensity of quantum dots (QDs), therefore, a rapid sensitive and selective fluorescence probe has been proposed for the detection of Cu²⁺ in aqueous solution. Under optimal conditions, the fluorescence intensity of CdTe/ZnS QDs was linearly proportional to the concentration of Cu²⁺ in the range from 2.5 × 10^–9 M to 17.5 × 10^–7 M with the limit of 1.5 × 10^–9 M and relative standard deviation of 0.23%. The quenching mechanism is static quenching with recoveries of 97.30–102.75%. Copyright © 2015 John Wiley & Sons, Ltd.     

ADSORPTION AND UPTAKE OF COPPER BY THE GREEN ALGA SCENEDESMUS SUBSPICATUS (CHLOROPHYTA)1

Copper (II) accumulation has been investigated in the green alga Scenedesmus subspicatus G. Brinkmann considering both adsorption and uptake kinetics. Experiments were conducted in a Cu- and PH-buffered medium at different free Cu²⁺ concentrations that were neither growth limiting nor toxic. We distinguished between adsorption on the cell surface and intracellular uptake by extracting copper from the cells with EDTA. Data from short-term experiments were compared with data obtained from experiments under steady state conditions. The accumulation of Cu can be described by two processes, an initial fast adsorption occurring within a minute followed by a slower intracellular uptake. Metal uptake followed Michaelis-Menten kinetics and is mediated by two systems, one with low and the other with high affinity. The maximum uptake rates (1.30 × 10^?-¹⁰ mol·[g dry wt algae]^?1· min^?1, 3.67 × 10^?-⁹ mol·[g dry wt algae]^?1·min^?1), and the half-saturation constants (6.84 × 10^?-¹⁴ M, 2.82 × 10^?-¹² M) for the two uptake systems were determined using the Lineweaver-Burk plot. The calculated maximum concentration of binding sites on the surface of the algae is initially higher (9.0 × 10^?-⁶ mol Cu.[g dry wt algae]^?1) than under steady state conditions (2.9 × 10^?-⁶ mol Cu·[g dry wt algae]^?1). This suggests that the initial binding to the algal surface comprises the binding to specific transport ligands as well as to inert adsorption sites. The conditional stability constant of the Cu binding to surface ligands was calculated as log K_Cu= 11.0 at pH 7.9. This freshwater alga has a high ability to accumulate Cu, reflecting its adaptation to the bioavailable concentration of copper.     

Comparative effects of adenine analogs upon metabolic cooperation between Chinese hamster cells with different levels of adenine phosphoribosyltransferase activity

Colony formation by variant Chinese hamster cells highly resistant to adenine analogs and deficient in adenine phosphoribosyltransferase (APRT) activity was measured after co-cultivation with APRT⁺, CHO-K1 cells in medium containing one of three different adenine analogs. Depending upon the density of APRT⁺ cells and the specific adenine analog, large differences in the recovery of APRT^? colonies were observed. The particular adenine analog and APRT⁺ cell density were more significant factors in the recovery of APRT^? colonies than the concentration of the analog or the level of APRT activity. The number of wild-type cells (CHO-K1) required to inhibit formation of APRT^? colonies by 50% (mean lethal density; MLD₅₀) with 65 μg/ml 8-aza-adenine (AzA) as the selective drug was 8.0 × 10⁵ cells/100 mm dish (1.5 × 10⁴/cm²). With 100 μg/ml 2,6-diaminopurine (DAP) the MLD₅₀ for CHO-K1 was 4.0 × 10⁵ cells/100 mm dish (7.3 × 10³/cm²). The MLD₅₀ for CHO-K1 when the DAP concentration was decreased to 50 μg/ml was only slightly higher, 5 × 10⁵ cells/100 mm dish (9.1 × 10³/cm²). The most toxic effect was observed with 2-fluoroadenine (FA). The MLD₅₀ for CHO-K1 in 2 μg/ml FA was 4.5 × 10⁴ cells/100 mm dish (8.2 × 10²/cm²), a cell density which permits minimal direct contact between APRT⁺ and APRT^? cells. The toxic effects of FA on individually resistant, APRT^? cells were found to be mediated by metabolites released into the medium by dying APRT⁺ cells. This metabolite toxicity to APRT^? cells was also demonstrated in mixtures with cells having only 8% of wild-type APRT activity. The MLD₅₀ for these APRT⁺ (8%) cells in 2 μg/ml FA was 7.5 × 10⁴ cells/100 dish (1.4 × 10³/cm²), a small difference from the MLD₅₀ for cells with wild-type levels of APRT activity. The differences in the recovery of APRT^? colonies from mixtures with APRT⁺ cells in these three adenine analogs are critical to the design of procedures for the selection of APRT^? cells from populations of APRT⁺ cells and emphasize the importance of establishing the parameters of metabolic cooperation, not only in terms of cell density but also with regard to the particular selective agent, in any experiment designed to determine precise mutation rates or to test putative mutagens upon mammalian cells in culture.