Chitosan Contribution to Therapeutic and Vaccinal Approaches for the Control of Leishmaniasis.

The control of leishmaniases, a complex parasitic disease caused by the protozoan parasite Leishmania, requires continuous innovation at the therapeutic and vaccination levels. Chitosan is a biocompatible polymer administrable via different routes and possessing numerous qualities to be used in the antileishmanial strategies. This review presents recent progress in chitosan research for antileishmanial applications. First data on the mechanism of action of chitosan revealed an optimal in vitro intrinsic activity at acidic pH, high-molecular-weight chitosan being the most efficient form, with an uptake by pinocytosis and an accumulation in the parasitophorous vacuole of Leishmania-infected macrophages. In addition, the immunomodulatory effect of chitosan is an added value both for the treatment of leishmaniasis and the development of innovative vaccines. The advances in chitosan chemistry allows pharmacomodulation on amine groups opening various opportunities for new polymers of different size, and physico-chemical properties adapted to the chosen routes of administration. Different formulations have been studied in experimental leishmaniasis models to cure visceral and cutaneous leishmaniasis, and chitosan can act as a booster through drug combinations with classical drugs, such as amphotericin B. The various architectural possibilities given by chitosan chemistry and pharmaceutical technology pave the way for promising further developments.

Ultrasmall AgNP-Impregnated Biocompatible Hydrogel with Highly Effective Biofilm Elimination Properties.

Ultrasmall silver nanoparticles (AgNPs; size < 3 nm) have attracted a great deal of interest as an alternative to commercially available antibiotics due to their ability to eliminate a wide range of microbial pathogens. However, most of these ultrasmall AgNPs are highly reactive and unstable, as well as susceptible to fast oxidation. Therefore, both the stability and toxicity remain major shortcomings for their clinical application and uptake. To circumvent these problems, we present a novel strategy to impregnate ultrasmall AgNPs into a biocompatible thermosensitive hydrogel that enables controlled release of silver alongside long-term storage stability and highly potent antibacterial activity. The advantage of this strategy lies in the combination of a homogenous dispersion of AgNPs in a hydrogel network, which serves as a sustained-release reservoir, and the unique feature of ultrasmall AgNP size, which provides an improved biofilm eradication capacity. The superior biofilm dispersion properties of the AgNP hydrogel is demonstrated in both single-species and multispecies biofilms, eradicating ∼80% of established biofilms compared to untreated controls. Notably, the effective antibacterial concentration of the formulation shows minimal toxicity to human fibroblasts and keratinocytes. These findings present a promising novel strategy for the development of AgNP hydrogels as an efficient antibacterial platform to combat resistant bacterial biofilms associated with wound infections.

Light-mediated effects of CdTe-MSA quantum dots on the autofluorescence of freshwater green microalgae: Spectroscopic studies.

The water-soluble semiconductor quantum dots (QDs) serve as optically detectable models of nanoparticles and are commonly applied as photoluminescent markers in biological systems. The unicellular algae represent a popular model system suitable to evaluate pollution-induced effects. There is growing experimental evidence that release of metal ions cannot account for potential toxicity of metal containing nanoparticles, however, the underlying mechanisms are not clearly understood. Surrounding environment and illumination conditions are among the most important factors affecting the stability of QDs as well as the interaction between nanoparticles and cells such as microalgae. The measurements of changes in photoluminescence (PL) of QDs and autofluorescence (AF) of microalgae can thus be used as a non-invasive screening method for detecting mutual effects of nanoparticles and algae cells on each other under natural conditions. In this study, CdTe quantum dots (a peak of PL at 550 nm) capped with a mercaptosuccinic acid (MSA) were introduced into aqueous ionic medium containing wild type green freshwater microalgae Scenedesmus and Chlorella sp. cells under artificial and natural ambient illumination. The spectroscopy and microscopy techniques were applied to observe both the influence of the microalgae on the spectral properties of negatively charged CdTe-MSA quantum dots and the effects of nanoparticles on the microalgae. The presence of algae cells revealed a protecting effect on both medium-dependent and radiation-induced changes in photoluminescence properties of QDs, which could be related with the increased stability of the capping layer. The effects on cellular AF intensity and the interaction of QDs with cellular surface depended on type of microalgae. The observed changes in AF spectral properties and AF induction signals can't be explained only by the photodegradation of QDs and have revealed the ability of nanoparticles to retard the photoadaptation of wild type microalgae under naturally varying illumination conditions.

Nitric oxide-loaded chitosan nanoparticles as an innovative antileishmanial platform.

Leishmaniasis is a neglected tropical disease that demands for new therapeutic strategies due to adverse side effects and resistance development promoted by current drugs. Nitric oxide (NO)-donors show potential to kill Leishmania spp. but their use is limited because of their instability. In this work, we synthesize, characterize, and encapsulate S-nitroso-mercaptosuccinic acid into chitosan nanoparticles (NONPs) and investigate their activity on promastigotes and intracellular amastigotes of Leishmania (Leishmania) amazonensis. Cytotoxicity on macrophages was also evaluated. We verified that NONPs reduced both forms of the parasite in a single treatment. We also noticed reduction of parasitophorous vacuoles as an evidence of inhibition of parasite growth and resolution of infection. No substantial cytotoxicity was detected on macrophages. NONPs were able to provide a sustained parasite killing for both L. (L.) amazonensis infective stages with no toxicity on macrophages, representing a promising nanoplatform for cutaneous leishmaniasis.

Magnetic metal-organic frameworks containing abundant carboxylic groups for highly effective enrichment of glycopeptides in breast cancer serum.

A mercaptosuccinic acid functionalized hydrophilic magnetic metal-organic framework nanocomposite (denoted as mMOF@Au-MSA) was proposed and synthesized to provide an excellent platform for glycopeptide analysis. The novel nanomaterial integrated favorable advantages such as robust magnetic response from Fe3O4 magnetic nanoparticles, large surface area contributed by MOF, abundant ultra-high hydrophilic carboxylic groups from mercaptosuccinic acid, as well as unbiased affinity toward different types of glycopeptides. This nanocomposite was successfully utilized to capture glycopeptides from standard protein digests with the high selectivity and great sensitivity of 0.5 fmol µL-1. Notably, 307 glycopeptides assigned to 96 glycoproteins were identified from only 2 µL serum of breast cancer patient. The satisfying achievement indicated that the as-prepared nanopartical had promising potential in exploring the knowledge of glycoproteins in breast cancer.

AIE-active self-assemblies from a catalyst-free thiol-yne click reaction and their utilization for biological imaging.

Aggregation-induced emission (AIE) should be the most interest fluorescent phenomenon over the past few decades. The luminescence polymeric nanoparticles (LPNs) with AIE characteristic have attracted great research attention for biological imaging and many other biomedical applications owing to their good biocompatibility and negative toxicity. However, the preparation of LPNs with desirable optical properties using traditional organic dyes still remains a great challenge for the aggregation-caused quenching (ACQ) effect and aggregation of hydrophobic dyes in the core of LPNs. In this work, we reported a novel and simple method for fabrication of biodegradable AIE-active LPNs via the combination of condensation and click reactions. For preparation of these AIE-active LPNs, the thiol groups-containing hydrophilic copolymers (PEG-MA) were first synthesized through the condensation reaction between polyethylene glycol and mercaptosuccinic acid. The PEG-MA copolymers were further reacted with AIE dye PhE-OE through a catalyst-free thiol-yne click reaction. These obtained PEG-MA-PhE LPNs were fully characterized by a number of characterization techniques. All the results confirmed that PEG-MA-PhE LPNs possess excellent compatibility, intense red luminescence, great photostability and high water dispersibility. These features make PEG-MA-PhE LPNs promising candidates for various biomedical applications.

Biodegradable crosslinked polyesters derived from thiomalic acid and S-nitrosothiol analogues for nitric oxide release.

Crosslinked polyesters with Young's moduli similar to that of certain soft biological tissues were prepared via bulk polycondensation of thiomalic acid and 1,8-octanediol alone, and with citric or maleic acid. The copolymers were converted to nitric oxide (NO)-releasing S-nitrosothiol (RSNO) analogues by reaction with tert-butyl nitrite. Additional conjugation steps were avoided by inclusion of the thiolated monomer during the polycondensation to permit thiol conversion to RSNOs. NO release at physiological pH and temperature (pH 7.4, 37 °C) was determined by chemiluminescence-based NO detection. The average total NO content for poly(thiomalic-co-maleic acid-co-1,8-octanediol), poly(thiomalic-co-citric acid-co-1,8-octanediol), and poly(thiomalic acid-co-1,8-octanediol) was 130 ± 39 µmol g-1, 200 ± 35 µmol g-1, and 130 ± 11 µmol g-1, respectively. The antibacterial properties of the S-nitrosated analogues were confirmed against Escherichia coli and Staphylococcus aureus. The hydrolytic degradation products were analyzed by time-of-flight mass spectrometry after a 10-week study to investigate their composition. Tensile mechanical tests were performed on the non-nitrosated polymers as well as their S-nitrosated derivatives and suggested that the materials have appropriate Young's moduli and elongation values for biomedical applications.

Antifungal graphene oxide-borneol composite.

Although antibacterial activities of graphene oxide (GO) and its derivatives have been investigated comprehensively, their antifungal properties are still less reported. Yet, fungal contamination seriously threatens the public health. Herein, we present a design of graphene oxide-borneol (GOB) composite, and report its great antifungal effect. This GOB composite is prepared by esterification of borneol with thiomalic-acid-modified GO sheets, where the linker molecule is used to increase surface carboxyl groups. As a result, the antifungal activity displays a dramatically conversion from no activity of GO and its derivatives to distinct antifungal adhesion and growth inhibition of the GOB. Under microscopy, few spores can be found on the GOB surface, while large numbers of sporangia and spores adhere and grow on the control groups. It is also worth noting that on the GOB sample the fallen spore does not germinate even after 5days, demonstrating a long-term antifungal effect of the GOB composite. Further studies confirm that carbon stereochemistry rather than wettability plays a crucial role on the antifungal adhesion properties. This study not only highlights a promising GOB composite as a candidate of graphene-based antifungal agent, but also provides us with in-depth understanding of the interactions between fungi and graphene-based materials.

Redox sensitive cationic pullulan for efficient gene transfection and drug retention in C6 glioma cells.

Thiolated cationic pullulan was synthesized by conjugating pullulan with polyethyleneimine (PEI) and mercaptosuccinic acid (MSA). The formed conjugate was oxidized to obtain disulfide linked cationic pullulan (PPMSS). PPMSS exhibited good buffering capacity and nanoplexes formulated were of size less than 150nm. Nanoplexes formed with PPMSS are redox sensitive and susceptible to reductive cleavage by dithiothreitol (DTT) ensuring the intracellular release of DNA. In vitro, cytotoxic evaluation studies of polymers in C6 cell lines established its non-toxic nature. The studies using endocytosis inhibitors revealed the uptake pathways of nanoplexes. Further, the plasmid and polymer tracking studies indicated the successful unpacking of DNA from the nanoplexes and its nuclear localization. The gene transfection efficiency was established by the p53 gene expression studies. Furthermore, the ability of the polymer to inhibit efflux pumping in cancer cells has also been elucidated in terms of P-gp inhibition studies and drug retention kinetics using the anticancer drug, doxorubicin (DOX). Our results also suggest that greater retention of DOX was accompanied by the reduction of disulfide linkage by a ubiquitous intracellular stimulus, glutathione. Thus simultaneous gene and drug delivery using redox sensitive cationic polymers may have a promising potential in cancer therapy.

Nitric oxide-releasing chitosan nanoparticles alleviate the effects of salt stress in maize plants.

Nitric oxide (NO) is a signaling molecule involved in plant response to various abiotic stresses. However, the application of NO donors in agriculture is hampered by the instability of these compounds. Despite the successful uses of NO-releasing nanoparticles for biomedical purposes and the variety of nanomaterials developed as carrier systems of agrochemicals, the potential applications of nanocarriers for NO delivery in plants have not yet been tested. Herein, we report the synthesis and characterization of chitosan nanoparticles (CS NPs) containing the NO donor S-nitroso-mercaptosuccinic acid (S-nitroso-MSA). The efficiency of these NO-releasing NPs in mitigating the deleterious effects of salinity on maize plants was compared to that of the non-encapsulated NO donor. The NPs were synthesized through ionotropic gelation process, and mercaptosuccinic acid (MSA), the NO donor precursor, was encapsulated into CS NPs (91.07% encapsulation efficiency). Free thiol groups of MSA-CS NPs were nitrosated, leading to S-nitroso-MSA-CS NPs (NO-releasing NPs). The incorporation of S-nitroso-MSA into CS NPs allowed a sustained NO release. Treatments of salt-stressed maize plants with S-nitroso-MSA-CS NPs resulted in a higher leaf S-nitrosothiols content compared to that of free S-nitroso-MSA. Moreover, S-nitroso-MSA-CS NPs were more efficient than was the free NO donor in the amelioration of the deleterious effects of salinity in photosystem II activity, chlorophyll content and growth of maize plants because the protective action of the nanoencapsulated S-nitroso-MSA was achieved at lower dosages. Overall, these results demonstrate the positive impact of S-nitroso-MSA nanoencapsulation in increasing NO bioactivity in maize plants under salt stress.

Colorimetric Detection of Cadmium Ions Using DL-Mercaptosuccinic Acid-Modified Gold Nanoparticles.

A colorimetric assay has been developed for detection of Cd²âº utilizing DL-mercaptosuccinic acid-modified gold nanoparticles (MSA-AuNPs). The method showed good selectivity for Cd²âº over other metal ions. As a result, the linear relationships (r > 0.9606) between concentration 0.07 mM and 0.20 mM for cadmium ion were obtained. The detection limit was as low as 0.07 mM by the naked eye. The effect of pH on the aggregation was optimized. The MSA-AuNPs probe could be used to detect Cd²âº in an aqueous solution based on the aggregation-induced color change of MSA-AuNPs.

Synthesis of porous starch xerogels modified with mercaptosuccinic acid to remove hazardous gardenia yellow.

Mercaptosuccinic acid (MSA) molecules were inserted into potato starch, leading to the breaking of intrinsic H-bonds within macromolecular chains of starch and the formation of intermolecular H-bonds between MSA and starch, which could be verified by Fourier transform infrared spectroscopy (FT-TR). MSA modified porous starch xerogels (PSX/MSA) were obtained after freeze-drying the MSA modified starch, and they were characterized by field emission scanning electron microscopy (FESEM), exhibiting the intriguing porous structure due to the separation of starch chains by MSA molecules. The PSX/MSA were then used as the adsorbents to remove gardenia yellow (GY), a natural colorant with genotoxicity. Due to the porous structure of PSX and the introduced carboxyl groups from MSA, the adsorption capacity of the PSX/MSA was much higher than that of the starch xerogels alone (SX). The adsorption behaviors of GY by the PSX/MSA fitted both the Freundlich isotherm model and the pseudo-second-order kinetic model, and the efficient adsorption of GY suggested that the PSX/MSA might be potential adsorbents for the removal of dyes from contaminated aquatic systems.

Cytotoxicity of CdTe quantum dots in human umbilical vein endothelial cells: the involvement of cellular uptake and induction of pro-apoptotic endoplasmic reticulum stress.

Cadmium telluride quantum dots (CdTe QDs) have been proposed to induce oxidative stress, which plays a crucial role in CdTe QDs-mediated mitochondrial-dependent apoptosis in human umbilical vein endothelial cells (HUVECs). However, the direct interactions of CdTe QDs with HUVECs and their potential impairment of other organelles like endoplasmic reticulum (ER) in HUVECs are poorly understood. In this study, we reported that the negatively charged CdTe QDs (-21.63±0.91 mV), with good dispersity and fluorescence stability, were rapidly internalized via endocytosis by HUVECs, as the notable internalization could be inhibited up to 95.52% by energy depletion (NaN3/deoxyglucose or low temperature). The endocytosis inhibitors (methyl-ß-cyclodextrin, genistein, sucrose, chlorpromazine, and colchicine) dramatically decreased the uptake of CdTe QDs by HUVECs, suggesting that both caveolae/raft- and clathrin-mediated endocytosis were involved in the endothelial uptake of CdTe QDs. Using immunocytochemistry, a striking overlap of the internalized CdTe QDs and ER marker was observed, which indicates that QDs may be transported to ER. The CdTe QDs also caused remarkable ER stress responses in HUVECs, confirmed by significant dilatation of ER cisternae, upregulation of ER stress markers GRP78/GRP94, and activation of protein kinase RNA-like ER kinase-eIF2α-activating transcription factor 4 pathway (including phosphorylation of both protein kinase RNA-like ER kinase and eIF2α and elevated level of activating transcription factor 4). CdTe QDs further promoted an increased C/EBP homologous protein expression, phosphorylation of c-JUN NH2-terminal kinase, and cleavage of ER-resident caspase-4, while the specific inhibitor (SP600125, Z-LEVD-fmk, or salubrinal) significantly attenuated QDs-triggered apoptosis, indicating that all three ER stress-mediated apoptosis pathways were activated and the direct participation of ER in the CdTe QDs-caused apoptotic cell death in HUVECs. Our findings provide important new insights into QDs toxicity and reveal potential cardiovascular risks for the future applications of QDs.

Short- and Long-Term Tracking of Anionic Ultrasmall Nanoparticles in Kidney.

While biodistribution of nanoparticles (NPs) has been widely studied at the organ level, relatively little is known about their disposition in organs at the cellular level, especially after long-term exposure. The kidney is regarded as the key organ for the clearance of ultrasmall NPs (<5.5 nm). However, recent studies indicate that NPs in this size range could accumulate in the kidney for extended times without urinary excretion. Using negatively charged quantum dots (QDs) (∼3.7 nm) as a model system, we examined the suborgan disposition of anionic ultrasmall NPs in the kidney at the cellular level after intravenous injection by multiphoton microscopy coupled with fluorescence lifetime imaging. Most of the NPs were initially distributed in the peritubular capillaries or glomerular arterioles after injection, whereas they passed through the fenestrated glomerular endothelium and were gradually taken up by mesangial cells up to 30 days after injection. Only trace amounts of anionic QDs could be detected in the urine, which could be attributed to the barrier of the anionic glomerular basement membrane preventing filtration of anionic QDs. In contrast, cationic QDs of similar size (∼5.67 nm) were found to be readily excreted into urine. This study thus highlights the importance of surface charge in determining renal clearance of ultrasmall NPs. It provides a framework for characterizing and predicting the subcellular disposition in organs and long-term targeting of other NPs, with a physiologically based kinetic model being subsequently developed to describe the suborgan kinetics of anionic ultrasmall NPs.

Simultaneous Effect of Thiolation and Carboxylation of Chitosan Particles Towards Mucoadhesive Oral Insulin Delivery Applications: An In Vitro and In Vivo Evaluation.

Thiomalyl chitosan (TCS), a pH sensitive thiolated chitosan derivative, was developed and investigated towards oral protein delivery application. Particles of z-average 364 ± 5.6 nm with a negative zeta potential of 14.4 mV was obtained by tripolyphosphate cross linking of TCS. The release of insulin from TCS particles was significantly restricted at pH 1.2 minimizing up to about < 10% in 3 hours. The permeation enhancement ratio was found to 13 times higher than the FD4 alone and was 1.6 times higher than the unmodified chitosan particles. The protein protective properties of the matrix were established in presence of pepsin and pancreatic enzymes. Confocal microscopy studies proved the tight junction opening of Caco-2 cells by these thiolated chitosan particles and the in vivo studies on diabetic rats established its potential towards oral peptide delivery with pharmacological availability (PA) of 1.5%. The significance of this work is to establish that, the presence of multiple functional groups having similar property in the same matrix can improve its suitability as a promising candidate for oral peptide delivery with improved release characteristics, mucoadhesion as well as protecting the insulin activity and enhancing the permeability across the intestinal wall.

Combined nitric oxide-releasing poly(vinyl alcohol) film/F127 hydrogel for accelerating wound healing.

Nitric oxide (NO) releasing biomaterials represent a potential strategy for use as active wound dressings capable of accelerating wound healing. Topical NO-releasing poly(vinyl alcohol) (PVA) films and Pluronic F127 hydrogels (F127) have already exhibited effective skin vasodilation and wound healing actions. In this study, we functionalized PVA films with SNO groups via esterification with a mixture of mercaptosucinic acid (MSA) and thiolactic acid (TLA) followed by S-nitrosation of the SH moieties. These films were combined with an underlying layer of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide), i.e., PEO-PPO-PEO (Pluronic F127) hydrogel and used for the topical treatment of skin lesions in an animal model. The mixed esterification of PVA with MSA and TLA led to chemically crosslinked PVA-SNO films with a high swelling capacity capable of spontaneously releasing NO. Real time NO-release measurements revealed that the hydrogel layer reduces the initial NO burst from the PVA-SNO films. We demonstrate that the combination of PVA-SNO films with F127 hydrogel accelerates wound contraction, decreases wound gap and cellular density and accelerates the inflammatory phase of the lesion. These results were reflected in an increase in myofibroblastic differentiation and collagen type III expression in the cicatricial tissue. Therefore, PVA-SNO films combined with F127 hydrogel may represent a new approach for active wound dressings capable of accelerating wound healing.

Passive tumor targeting and imaging by using mercaptosuccinic acid-coated near-infrared quantum dots.

In this paper, we demonstrate the preparation of monodispersed quantum dots (QDs) as near-infrared (NIR) optical probes for in vivo pancreatic cancer targeting and imaging. The design of these luminescent probes involves functionalizing NIR QDs with ligand mercaptosuccinic acid (MSA), which targets the tumor site by enhanced permeability and retention effect. The colloidal and optical stability of the QDs can be maintained for >1 week. In vivo optical imaging studies in nude mice bearing pancreatic tumor show that the probes accumulate at tumor sites for >2.5 hours following intravenous injection of the functionalized NIR QDs. Tumor-labeling studies showed no evidence of harmful effects on the treated animals, even at a dose as high a ~50 mg/kg. These results demonstrate that the engineered MSA-functionalized QDs can serve as a diagnostic platform for early detection of cancer, as well as in image-guided precise surgical resection of tumors.

CdTe quantum dots conjugated to concanavalin A as potential fluorescent molecular probes for saccharides detection in Candida albicans.

Semiconductor colloidal quantum dots (QDs) have been applied in biological analysis due to their unique optical properties and their versatility to be conjugated to biomolecules, such as lectins and antibodies, acquiring specificity to label a variety of targets. Concanavalin A (Con A) lectin binds specifically to α-d-mannose and α-d-glucose regions of saccharides that are usually expressed on membranes of mammalian cells and on cell walls of microbials. Candida albicans is the most common fungal opportunistic pathogen present in humans. Therefore, in this work, this fungus was chosen as a model for understanding cells and biofilm-forming organisms. Here, we report an efficient bioconjugation process to bind CdTe (Cadmium Telluride) QDs to Con A, and applied the bioconjugates to label saccharide structures on the cellular surface of C. albicans suspensions and biofilms. By accomplishing hemagglutination experiments and circular dichroism, we observed that the Con A structure and biochemical properties were preserved after the bioconjugation. Fluorescence microscopy images of yeasts and hyphae cells, as well as biofilms, incubated with QDs-(Con A) showed a bright orange fluorescence profile, indicating that the cell walls were specifically labeled. Furthermore, flow cytometry measurements confirmed that over 93% of the yeast cells were successfully labeled by QD-(Con A) complex. In contrast, non-conjugated QDs or QDs-(inhibited Con A) do not label any kind of biological system tested, indicating that the bioconjugation was specific and efficient. The staining pattern of the cells and biofilms demonstrate that QDs were effectively bioconjugated to Con A with specific labeling of saccharide-rich structures on C. albicans. Consequently, this work opens new possibilities to monitor glucose and mannose molecules through fluorescence techniques, which can help to optimize phototherapy protocols for this kind of fungus.

Sensitive and selective colorimetric detection of Cu(2+) in aqueous medium via aggregation of thiomalic acid functionalized Ag nanoparticles.

A simple and effective colorimetric method for determination of Cu(2+) in real samples was developed. In this method, thiomalic acid functionalized silver nanoparticles (TMA-AgNPs) were prepared and changes in solution color, induced by the aggregation of TMA-AgNPs in the presence of Cu(2+), were employed for quantitative analysis. The surface plasmon resonance (SPR) band of our synthesized TMA-AgNPs was located at 392 nm and shifted to a longer wavelength after aggregation due to the interactions between carboxylate and Cu(2+). A band intensity ratio of A455/(A392-A455) was constructed and used to correlate with the concentration of Cu(2+). A linear relationship was found with a linear response up to 50 nM of Cu(2+). Due to the formation of a stable carboxylate Cu(2+) complex, highly sensitive detection of Cu(2+) was achieved with the estimated detection limit approaching 1 nM. Moreover, the formation of the stable complex leads to high selectivity in the detection of Cu(2+), which was verified by examination of 12 other metal ions. In the detection of Cu(2+) in real samples, results indicated that our proposed method is simple, sensitive and selective for application in such measurements.

Mercaptosuccinate dioxygenase, a cysteine dioxygenase homologue, from Variovorax paradoxus strain B4 is the key enzyme of mercaptosuccinate degradation.

The versatile thiol mercaptosuccinate has a wide range of applications, e.g. in quantum dot research or in bioimaging. Its metabolism is investigated in Variovorax paradoxus strain B4, which can utilize this compound as the sole source of carbon and sulfur. Proteomic studies of strain B4 resulted in the identification of a putative mercaptosuccinate dioxygenase, a cysteine dioxygenase homologue, possibly representing the key enzyme in the degradation of mercaptosuccinate. Therefore, the putative mercaptosuccinate dioxygenase was heterologously expressed, purified, and characterized in this study. The results clearly demonstrated that the enzyme utilizes mercaptosuccinate with concomitant consumption of oxygen. Thus, the enzyme is designated as mercaptosuccinate dioxygenase. Succinate and sulfite were verified as the final reaction products. The enzyme showed an apparent Km of 0.4 mM, and a specific activity (Vmax) of 20.0 µmol min(-1) mg(-1) corresponding to a kcat of 7.7 s(-1). Furthermore, the enzyme was highly specific for mercaptosuccinate, no activity was observed with cysteine, dithiothreitol, 2-mercaptoethanol, and 3-mercaptopropionate. These structurally related thiols did not have an inhibitory effect either. Fe(II) could clearly be identified as metal cofactor of the mercaptosuccinate dioxygenase with a content of 0.6 mol of Fe(II)/mol of enzyme. The recently proposed hypothesis for the degradation pathway of mercaptosuccinate based on proteome analyses could be strengthened in the present study. (i) Mercaptosuccinate is first converted to sulfinosuccinate by this mercaptosuccinate dioxygenase; (ii) sulfinosuccinate is spontaneously desulfinated to succinate and sulfite; and (iii) whereas succinate enters the central metabolism, sulfite is detoxified by the previously identified putative molybdopterin oxidoreductase.