Sulfhydryl Moiety Research Articles

Mercury toxicity resulting from enzyme alterations- minireview.

Mercury is widely known for its detrimental effects on living organisms, whether in its elemental or bonded states. Recent comparative studies have shed light on the biochemical implications of mercury ingestion, both in low, persistent concentrations and in elevated acute dosages. Studies have presented models that elucidate how mercury disrupts healthy cells. Mercury's unique ability to interfere with crucial enzymatic processes at deposition sites is a vital feature of these models. The strong affinity for the sulfhydryl moieties of enzyme catalytic sites leads to enzyme inactivation through permanent covalent modifications. This inactivation can have catastrophic effects on an organism's metabolic functions. Moreover, it has been found that mercury's binding to sulfhydryl moieties is highly nonspecific and can occur in various ways. This review aimed to explore the effects of mercury on a broad spectrum of enzymes with a specific focus on how these alterations can detrimentally affect several metabolic pathways.

Imaging Very Late Antigen-4 on MOLT4 Leukemia Tumors with Cysteine Site-Specific 89Zr-Labeled Natalizumab Immuno-Positron Emission Tomography.

Very late antigen-4 (VLA4; CD49d) is a promising immune therapy target in treatment-resistant leukemia and multiple myeloma, and there is growing interest in repurposing the humanized monoclonal antibody (Ab), natalizumab, for this purpose. Positron emission tomography with radiolabeled Abs (immuno-PET) could facilitate this effort by providing information on natalizumab's in vivo pharmacokinetic and target delivery properties. In this study, we labeled natalizumab with 89Zr specifically on sulfhydryl moieties via maleimide-deferoxamine conjugation. High VLA4-expressing MOLT4 human T cell acute lymphoblastic leukemia cells showed specific 89Zr-natalizumab binding that was markedly blocked by excess Ab. In nude mice bearing MOLT4 tumors, 89Zr-natalizumab PET showed high-contrast tumor uptake at 7 days postinjection. Biodistribution studies confirmed that uptake was the highest in MOLT4 tumors (2.22 ± 0.41%ID/g) and the liver (2.33 ± 0.76%ID/g), followed by the spleen (1.51 ± 0.42%ID/g), while blood activity was lower at 1.12 ± 0.21%ID/g. VLA4-specific targeting in vivo was confirmed by a 58.1% suppression of tumor uptake (0.93 ± 0.15%ID/g) when excess Ab was injected 1 h earlier. In cultured MOLT4 cells, short-term 3 day exposure to the proteasome inhibitor bortezomib (BTZ) did not affect the α4 integrin level, but BTZ-resistant cells that survived the treatment showed increased α4 integrin expression. When the effects of BTZ treatment were tested in mice, there was no change of the α4 integrin level or 89Zr-natalizumab uptake in MOLT4 leukemia tumors, which underscores the complexity of tumor VLA4 regulation in vivo. In conclusion, 89Zr-natalizumab PET may be useful for noninvasive monitoring of tumor VLA4 and may assist in a more rational application of Ab-based therapies for hematologic malignancies.

TDDFT study on a fluorescent probe for distinguishing analogous thiols based on smiles rearrangement

The complete excited-state sensing mechanism of a fluorescent probe capable of distinguishing cysteine/homocysteine and glutathione from analogous biological thiols has been investigated. Using a TDDFT method, the nature of the fluorescence differences in the detection of thiols by the probe has been explained at the molecular level. Calculation results imply that the probe undergoes photoinduced electron transfer (PET) from the fluorophore to the nitrobenzooxadiazole (NBD)-based acceptor in the excited state. In the presence of a thiol, the NBD moiety is cleaved and the red fluorescence emission of the fluorophore is enhanced through inhibition of the PET process. The sulfur-substituted NBD-thiol product is predisposed to undergo excited-state torsion, leading to fluorescence quenching. However, for cysteine and homocysteine, their appropriate distances lead to Smiles rearrangements with relatively low activation energies (26.60 kJ/mol and 42.94 kJ/mol, respectively) and the emission of a distinct green fluorescence at ambient temperature. It has been theoretically confirmed that the distance between two reactive sites, such as sulfhydryl and amino moieties, can be used to distinguish different thiols, thus providing rational support for the control of fluorescence activity and the design of probe molecules.

Bromelain: a review of its mechanisms, pharmacological effects and potential applications.

The utilization of plant-derived supplements for disease prevention and treatment has long been recognized because of their remarkable potential. Ananas comosus, commonly known as pineapple, produces a group of enzymes called bromelain, which contains sulfhydryl moieties. Recent studies have shown that bromelain exhibits a wide range of activities, including anti-inflammatory, anti-diabetic, anti-cancer, and anti-rheumatic properties. These properties make bromelain a promising drug candidate for the treatment of various diseases. The anti-inflammatory activity of bromelain has been shown to be useful in treating inflammatory conditions such as osteoarthritis, rheumatoid arthritis, and asthma, whereas the anti-cancer activity of bromelain is via induction of apoptosis, inhibition of angiogenesis, and enhancement of the body's immune response. The anti-diabetic property of bromelain is owing to the improvement in glucose metabolism and reduction in insulin resistance. The therapeutic potential of bromelain has been investigated in numerous preclinical and clinical studies and a number of patents have been granted to date. Various formulations and delivery systems are being developed in order to improve the efficacy and safety of this molecule, including the microencapsulated form to treat oral inflammatory conditions and liposomal formulations to treat cancer. The development of novel drug delivery systems and formulations has further ameliorated the therapeutic potential of bromelain by improving its bioavailability and stability, while reducing the side effects. This review intends to discuss various properties and therapeutic applications of bromelain, along with its possible mechanism of action in treating various diseases. Recent patents and clinical trials concerning bromelain have also been covered.

Cysteine-specific 89Zr-labeled anti-CD25 IgG allows immuno-PET imaging of interleukin-2 receptor-α on T cell lymphomas.

Positron emission tomography (PET) using radiolabeled Abs as imaging tracer is called immuno-PET. Immuno-PET can verify therapeutic Ab delivery and can noninvasively quantify global levels of target expression in tumors of living subjects. The interleukin-2 receptor α chain (IL-2Rα; CD25) is a promising target for immune therapy and radioimmunotherapy of lymphomas. Immuno-PET could facilitate this approach by visualizing CD25 expression in vivo. We prepared 89Zr-anti-CD25 IgG specifically labeled to sulfhydryl moieties by maleimide-deferoxamine conjugation. CD25(+) SUDHL1 human T-cell lymphoma cells showed high anti-human 89Zr-CD25 IgG binding that reached 32-fold of that of CD25(-) human lymphoma cells and was completely blocked by excess unlabeled Ab. In SUDHL1 tumor-bearing nude mice, pharmacokinetic studies demonstrated exponential reductions of whole blood and plasma activity following intravenous 89Zr-anti-CD25 IgG injection, with half-lives of 26.0 and 23.3 h, respectively. SUDHL1 tumor uptake of 89Zr-CD25 IgG was lower per weight in larger tumors, but blood activity did not correlate with tumor size or blood level of human CD25, indicating minimal influence by circulating soluble CD25 protein secreted from the lymphoma cells. 89Zr-CD25 IgG PET allowed high-contrast SUDHL1 lymphoma visualization at five days. Biodistribution studies confirmed high tumor 89Zr-CD25 IgG uptake (8.7 ± 0.9%ID/g) that was greater than blood (5.2 ± 1.6%ID/g) and organ uptakes (0.7 to 3.5%ID/g). Tumor CD25-specific targeting was confirmed by suppression of tumor uptake to 4.3 ± 0.2%ID by excess unlabeled CD25 IgG, as well as by low tumor uptake of 89Zr-labeled IgG2a isotype control Ab (3.6 ± 0.9%ID). Unlike CD25(+) lymphocytes from mouse thymus that showed specific uptake of anti-mouse 89Zr-CD25 IgG, EL4 mouse lymphoma cells had low CD25 expression and showed low uptake. In immunocompetent mice bearing EL4 tumors, anti-mouse 89Zr-CD25 IgG displayed low uptakes in normal organs as well as in the tumor. Furthermore, the biodistribution was not influenced by Ab blocking, indicating that specific uptake in nontumor tissues was minimal. 89Zr-CD25 IgG immuno-PET may thus be useful for imaging of T-cell lymphomas and noninvasive assessment of CD25 expression on target cells in vivo.

Interactions and toxicity of non-essential heavy metals (Cd, Pb and Hg): lessons from Drosophila melanogaster.

Some heavy metals are essential in trace amounts, enhancing enzyme functioning and other intracellular molecules. Others are explicitly toxic at low concentrations, increasing the risk of organ-related toxicity. Non-essential metals have similar mechanisms of toxicity to essential metals. These include the modifiable change in oxidation states, interaction with sulfhydryl moieties of proteins and indirect modification of nucleic acids. Ultimately, oxidative stress is generated, and potentiation of damage ensues. The susceptibility, sensitivity, genetic resources, and cellular response of Drosophila melanogaster to heavy metal exposure and toxicity have made this insect appropriate for toxicological studies. In this review, we focus on the toxicological impacts of non-essential metals (Cd, Pb, and Hg) in Drosophila and discuss its cellular and developmental responses to increasing concentrations of these metals. We also suggest current or proposed therapeutic alternatives, as well as dimensions that may improve the studies of non-essential metal biology.

Strategies for the surface biofunctionalization of sulfur-based chalcogenide infrared optical glasses

Effective surface functionalization plays a critical role in the selectivity and sensitivity of sensing devices. While chalcogenide glasses (ChG) have received a lot of attention as promising optical materials for mid-IR biosensors, there have been just a few reported instances of their successful (bio)functionalization. In the present work, we explore three different approaches for the grafting of heterobifunctional derivatives on Ge25Sb10S65 substrates. One of these approaches consisted in the use of traditional silane chemistry to functionalize hydroxyl moieties on the ChG, while the other two are novel strategies based on the potential reactivity of maleimide and cyclooctyne derivatives for sulfhydryl moieties at the surface of the inorganic glass matrix. Using these approaches, biotinylated surfaces were prepared and the selective grafting of a fluorescent-labeled protein was then validated via IR spectroscopy, AFM and fluorescence. Our results showed that successful biofunctionalization of Ge-Sb-S ChG was achieved by all three methods. Moreover, the functionalization process can be easily adapted for substrates with any type of geometry and for the grafting of different molecules at the surface, paving the way for the future development of infrared optical sensing systems exhibiting surface bio selectivity.

Siarkowodór — od kłopotliwego zapachu do podręcznika farmakologii

The history of hydrogen sulphide (H 2 S), which is a toxic, colourless gas with peculiar, creepy smell of rotten eggs is long. It’s critical role not only in development of the Universe but also in evolution of living organisms has been well documented. Apart from the contribution in evolution the hydrogen sulphide has been recognized as an important gasotransmitter in numerous biological processes. In the paper we review recent discoveries on dominating pathways leading to synthesis of endogenous hydrogen sulphide, we summarize the key biological effects of hydrogen sulphide of importance for cardiovascular homeostasis. Further, we point out selected molecules of natural origin and also products of chemical synthesis known to increase via different mechanisms the bioavailability of hydrogen sulphide. Among drugs approved for human pharmacotherapy zofenopril is the one shown to possess such an ability. The drug belongs to angiotensin-converting enzyme inhibitors (ACEIs) having sulfhydryl moiety in its chemical structure. One of the metabolite of zofenopril was shown to increase the expression of enzyme synthesizing hydrogen sulphide and to elevate both plasma and myocardial levels of hydrogen sulphide. The above mentioned effects have not been shown by using other ACEI. Hydrogen sulphide realising ability of zofenopril apart of its long-term inhibition of ACEIs may be considered as potential explanation for prognostic advantage over other drugs of the same class shown in prospective randomised trial.

The tea catechin epigallocatechin gallate inhibits NF-κB-mediated transcriptional activation by covalent modification

Potential health benefits of consuming tea are thought to include anti-inflammatory actions of its constituent flavonoids including catechins, which are well-recognized antioxidants. We analyzed and discovered a novel mechanism by which epigallocatechin gallate (EGCG), the most abundant polyphenol in tea and a putative health-promoting constituent, inhibits activation of the nuclear transcription factor NF-κB, which mediates inflammatory responses to cytokines and other agents. We found that EGCG inhibits NF-κB-p65 transcriptional activity, by preventing NF-κB-p65 binding to κBs in normal human bronchial epithelial cells. We also analyzed the chemical mechanism by which EGCG binds directly to NF-κB-p65, and found that it involves covalent reaction via enones within EGCG ring structures, as the oxidizer diamide, which prevents 1, 4-addition reactions, blocked adduct-forming reaction of biotinylated EGCG with NF-κB-p65. Such blockade was inhibited by competing unlabeled EGCG. Furthermore, such covalent binding reflected irreversible reaction of EGCG with sulfhydryls of NF-κB-p65, as it was inhibited by glutathione but not reversible by it. We identified the reactive sulfhydryl moiety as that of cysteine, as S-carboxymethylation to block cysteine sulfhydryls prevented NF-κB-p65-Cys-alkylation reaction with EGCG. We also tested if EGCG can inhibit NF-κB-p65 binding to DNA within the nucleus, after its phosphorylation and translocation (activation). EGCG did not alter intranuclear phosphorylation levels of NF-κB-p65, but strongly repressed DNA-binding ability of activated NF-κB-p65, indicating that EGCG inhibits NF-κB-p65 DNA binding activity even without altering NF-κB-p65 phosphorylation or expression. These findings thus reveal a novel mechanism by which EGCG inhibits transcriptional activity of NF-κB-p65, that may potentially contribute to anti-inflammatory and health-promoting effects of EGCG and consumption of tea.

Development of a water-soluble near-infrared fluorescent probe for endogenous cysteine imaging.

We designed and synthesized a water-soluble near-infrared (NIR) fluorescent probe with the recognition unit of the cyanine-like structure and acrylate group. Through an aromatic ring nucleophilic substitution reaction based on sulfhydryl moiety, an off-on fluorescence response toward cysteine (Cys) was realized. The probe exhibited excellent spectral performance with an emission wavelength of 720nm and a detection limit of 0.20μM. The spectral properties, selectivity and anti-interference performance of the probe were systematically investigated. Density functional theory (DFT) calculations were conducted to clarify the luminescence mechanism of the probe. Furthermore, the probe was successfully applied to the detection of free Cys in human serum and the NIR imaging of endogenous Cys in living cells. Thus, the probe has a promising application prospect in clinical diagnosis and fluorescence imaging.

The effect of metal loading on bacterial Hg adsorption

The mechanism of metal adsorption onto bacteria can change as a function of metal loading of the binding sites, and therefore the mechanisms of adsorption and the thermodynamic stability constants for the important bacterial surface complexes must be determined in order to accurately model metal behavior in near-surface geologic systems. In this study, batch metal adsorption experiments were conducted as a function of pH from 4 to 8, and Hg loading from 10 to 200 μmol Hg/g (wet weight bacteria). The adsorption data are in poor agreement with predictions of the extent of Hg adsorption that used previously published Hg-bacteria stability constants generated from high metal loading experiments. The extent that the thermodynamic model underpredicts the measured Hg adsorption increases with decreasing Hg loading and pH, suggesting that the mechanism of adsorption changes as a function of Hg loading and that an additional Hg-bacterial surface complex becomes important under low, <200 μmol/g, Hg loading and low, <6.5, pH conditions. The new Hg-bacterial surface complex involves a high affinity, low abundance site, which is likely a sulfhydryl moiety based on x-ray absorption spectroscopy, and is important only under low Hg loading conditions. We model the enhanced adsorption onto this high affinity site using an additional Hg-bacterial surface complex to those considered in the model derived from the high Hg loading data, and we use our data to constrain values of the stability constants for this new complex. Our modeling results provide reasonable fits to the data, and the proposed set of stability constants should improve the accuracy of quantitative models of Hg complexation with bacterial surfaces as a function of Hg loading and pH.

The Effect of Single and Multiple SERAT Mutants on Serine and Sulfur Metabolism.

The gene family of serine acetyltransferases (SERATs) constitutes an interface between the plant pathways of serine and sulfur metabolism. SERATs provide the activated precursor, O-acetylserine for the fixation of reduced sulfur into cysteine by exchanging the serine hydroxyl moiety by a sulfhydryl moiety, and subsequently all organic compounds containing reduced sulfur moieties. We investigate here, how manipulation of the SERAT interface results in metabolic alterations upstream or downstream of this boundary and the extent to which the five SERAT isoforms exert an effect on the coupled system, respectively. Serine is synthesized through three distinct pathways while cysteine biosynthesis is distributed over the three compartments cytosol, mitochondria, and plastids. As the respective mutants are viable, all necessary metabolites can obviously cross various membrane systems to compensate what would otherwise constitute a lethal failure in cysteine biosynthesis. Furthermore, given that cysteine serves as precursor for multiple pathways, cysteine biosynthesis is highly regulated at both, the enzyme and the expression level. In this study, metabolite profiles of a mutant series of the SERAT gene family displayed that levels of the downstream metabolites in sulfur metabolism were affected in correlation with the reduction levels of SERAT activities and the growth phenotypes, while levels of the upstream metabolites in serine metabolism were unchanged in the serat mutants compared to wild-type plants. These results suggest that despite of the fact that the two metabolic pathways are directly connected, there seems to be no causal link in metabolic alterations. This might be caused by the difference of their pool sizes or the tight regulation by homeostatic mechanisms that control the metabolite concentration in plant cells. Additionally, growth conditions exerted an influence on metabolic compositions.

Evaluation of dermal adhesive formulations for topical application

Topical delivery is one of the main challenges in the pharmaceutical technology. This study aimed to synthesize a potential adhesive polymeric excipient stable enough for pronounced skin adhesiveness to pave the skin delivery pathway. Polyacrylic acid an anionic well established polymer in the pharmaceutical field was chemically modified with sulfhydryl moieties and in a second step, this sulfhydryl bearing polymer was prone to a preactivation process. During this process, mercaptonicotinic acid was covalently bound to the sulfhydryl groups of polyacrylic acid. The obtained polymeric conjugates were characterized with respect to viscosity measurements, and evaluated in terms of skin adhesive properties with the help of tensile strength assay as well as bond strength assay. Findings exhibited promising potential of newly synthesized preactivated polyacrylic acid in terms of adhesive properties. Preactivated polyacrylic acid revealed a 15.39-fold improved adhesion compared to unmodified polymeric excipient. Therefore, the herein designed preactivated polyacrylic acid shows interesting features for skin application such as scar and wound management.

A Critical Role of Ser26 Hydrogen Bonding in Aβ42 Assembly and Toxicity.

Amyloid β-protein (Aβ) assembly is a seminal process in Alzheimer's disease. Elucidating the mechanistic features of this process is thought to be vital for the design and targeting of therapeutic agents. Computational studies of the most pathologic form of Aβ, the 42-residue Aβ42 peptide, have suggested that hydrogen bonding involving Ser26 may be particularly important in organizing a monomer folding nucleus and in subsequent peptide assembly. To study this question, we experimentally determined structure-activity relationships among Aβ42 peptides in which Ser26 was replaced with Gly, Ala, α-aminobutryic acid (Abu), or Cys. We observed that aliphatic substitutions (Ala and Abu) produced substantially increased rates of formation of β-sheet, hydrophobic surface, and fibrils, and higher levels of cellular toxicity. Replacement of the Ser hydroxyl group with a sulfhydryl moiety (Cys) did not have these effects. Instead, this peptide behaved like native Aβ42, even though the hydropathy of Cys was similar to that of Abu and very different from that of Ser. We conclude that H bonding of Ser26 is the factor most important in its contribution to Aβ42 conformation, assembly, and subsequent toxicity.

Self‐assembling chitosan hydrogel: A drug‐delivery device enabling the sustained release of proteins

ABSTRACTThe development of a self‐assembling hydrogel, prepared from maleimide‐modified and thiolated chitosan (CS), is described. Under mild reaction conditions, the natural CS polymer was coupled with either maleimide or sulfhydryl moieties in a one‐step synthesis. Subsequently, these CS polymers spontaneously formed a covalently crosslinked CS hydrogel when mixed. The three‐dimensional network structure was visualized with scanning electron microscopy. The swelling and degradation behavior was evaluated, and viscosity measurements were conducted. The gel was loaded with the model protein albumin, and prolonged release was achieved. These properties were preserved after lyophilization and rehydration. This makes the hydrogel a promising scaffold for biological wound dressings for the treatment of chronic wounds. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018, 135, 45638.

Insulin autoimmune syndrome: a rare cause of hypoglycemia. The case report of the syndrome in pediatric practice

Insulin autoimmune syndrome (IAS) is characterized by spontaneous hypoglycemia accompanied by an increased insulin level and presence of insulin autoantibodies while no exogenous insulin was used. This disorder is the third leading cause of hypoglycemia in Southeastern Asia. The disorder develops in individuals with a genetic predisposition, mostly after they had administered drugs containing a sulfhydryl moiety, such as methimazole, penicillin g, etc. The syndrome is typical of adults and extremely rare among children. We report the case of developing IAS in a 3.5-year-old Caucasian girl, possibly induced by pyritinol therapy.

Synthesis of l-cysteine derivatives containing stable sulfur isotopes and application of this synthesis to reactive sulfur metabolome

Cysteine persulfide is an L-cysteine derivative having one additional sulfur atom bound to a cysteinyl thiol group, and it serves as a reactive sulfur species that regulates redox homeostasis in cells. Here, we describe a rapid and efficient method of synthesis of L-cysteine derivatives containing isotopic sulfur atoms and application of this method to a reactive sulfur metabolome. We used bacterial cysteine syntheses to incorporate isotopic sulfur atoms into the sulfhydryl moiety of L-cysteine. We cloned three cysteine synthases—CysE, CysK, and CysM—from the Gram-negative bacterium Salmonella enterica serovar Typhimurium LT2, and we generated their recombinant enzymes. We synthesized 34S-labeled L-cysteine from O-acetyl-L-serine and 34S-labeled sodium sulfide as substrates for the CysK or CysM reactions. Isotopic labeling of L-cysteine at both sulfur (34S) and nitrogen (15N) atoms was also achieved by performing enzyme reactions with 15N-labeled L-serine, acetyl-CoA, and 34S-labeled sodium sulfide in the presence of CysE and CysK. The present enzyme systems can be applied to syntheses of a series of L-cysteine derivatives including L-cystine, L-cystine persulfide, S-sulfo-L-cysteine, L-cysteine sulfonate, and L-selenocystine. We also prepared 34S-labeled N-acetyl-L-cysteine (NAC) by incubating 34S-labeled L-cysteine with acetyl coenzyme A in test tubes. Tandem mass spectrometric identification of low-molecular-weight thiols after monobromobimane derivatization revealed the endogenous occurrence of NAC in the cultured mammalian cells such as HeLa cells and J774.1 cells. Furthermore, we successfully demonstrated, by using 34S-labeled NAC, metabolic conversion of NAC to glutathione and its persulfide, via intermediate formation of L-cysteine, in the cells. The approach using isotopic sulfur labeling combined with mass spectrometry may thus contribute to greater understanding of reactive sulfur metabolome and redox biology.

Captopril/enalapril inhibit promiscuous esterase activity of carbonic anhydrase at micromolar concentrations: An in vitro study

The inhibitory activity of captopril, a thiol-containing competitive inhibitor of the angiotensin-converting enzyme, ACE, against esterase activity of carbonic anhydrase, CA was investigated. This small molecule, as well as enalapril, was selected in order to represents both thiol and carboxylate, as two well-known metal binding functional groups of metalloprotein inhibitors. Since captopril, has also been observed to inhibit other metalloenzymes such as tyrosinase and metallo-beta lactamase through binding to the catalytic metal ions and regarding CA as a zinc-containing metallo-enzyme, in the current study, we set out to determine whether captopril/enalapril inhibit CA esterase activity of the purified human CA II or not? Then, we revealed the inhibitors' potencies (IC50, Ki and Kdiss values) and also mode of inhibition. Our results also showed that enalapril is more potent CA inhibitor than captopril. Since enalapril represents no sulfhydryl moiety, thus carboxylate group may have a determinant role in inhibiting of CA esterase activity, the conclusion confirmed by molecular docking studies. Additionally, since CA inhibitory potencies of captopril/enalapril were much lower than those of classic sulfonamide drugs, the findings of the current study may explain why these drugs exhibit no effective CA inhibition at the concentrations reached in vivo and also may shed light on the way of generating new class of inhibitors that will discriminately inhibit various CA isoforms.

Mini-review: toxicity of mercury as a consequence of enzyme alteration.

Mercury, in both its elemental and bonded states, is noted for its negative effects on biological organisms. Recent anthropogenic and environmental disasters have spurred numerous comparative studies. These studies attempted to detail the biochemical implications of mercury ingestion, in low, persistent concentrations as well as elevated acute dosages. The studies propose models for mercuric action on healthy cells; which is centered on the element's disruption of key enzymatic processes at deposition sites. Mercury's high affinity for the sulfhydryl moieties of enzyme catalytic sites is a common motif for enzyme inactivation. These permanent covalent modifications inactivate the enzyme, thereby inducing devastating effects on an organism's metabolic functions. Mercury has been shown to be highly nonspecific in its binding to sulfhydryl moieties, and highly varied in terms of how it is encountered by living organisms. This review focuses on mercury's effects on a wide swath of enzymes, with emphasis on how these alterations deleteriously affect several metabolic pathways.

Broadly Applicable Strategy for the Fluorescence Based Detection and Differentiation of Glutathione and Cysteine/Homocysteine: Demonstration in Vitro and in Vivo.

Glutathione (GSH), cysteine (Cys), and homocysteine (Hcy) are small biomolecular thiols that are present in all cells and extracellular fluids of healthy mammals. It is well-known that each plays a separate, critically important role in human physiology and that abnormal levels of each are predictive of a variety of different disease states. Although a number of fluorescence-based methods have been developed that can detect biomolecules that contain sulfhydryl moieties, few are able to differentiate between GSH and Cys/Hcy. In this report, we demonstrate a broadly applicable approach for the design of fluorescent probes that can achieve this goal. The strategy we employ is to conjugate a fluorescence-quenching 7-nitro-2,1,3-benzoxadiazole (NBD) moiety to a selected fluorophore (Dye) through a sulfhydryl-labile ether linkage to afford nonfluorescent NBD-O-Dye. In the presence of GSH or Cys/Hcy, the ether bond is cleaved with the concomitant generation of both a nonfluorescent NBD-S-R derivative and a fluorescent dye having a characteristic intense emission band (B1). In the special case of Cys/Hcy, the NBD-S-Cys/Hcy cleavage product can undergo a further, rapid, intramolecular Smiles rearrangement to form a new, highly fluorescent NBD-N-Cys/Hcy compound (band B2); because of geometrical constraints, the GSH derived NBD-S-GSH derivative cannot undergo a Smiles rearrangement. Thus, the presence of a single B1 or double B1 + B2 signature can be used to detect and differentiate GSH from Cys/Hcy, respectively. We demonstrate the broad applicability of our approach by including in our studies members of the Flavone, Bodipy, and Coumarin dye families. Particularly, single excitation wavelength could be applied for the probe NBD-OF in the detection of GSH over Cys/Hcy in both aqueous solution and living cells.