Concentration Of Free Zn2 Research Articles

Synthesis of ZnO Crystal with Micro‐Nano Structures by a Continuous Co‐Precipitation Method: Morphological Evolution and Mechanism Analysis

AbstractZnO crystals are employed in numerous industries, but precisely controlling their morphologies is still challenging. In this work, a continuous flow process for synthesizing ZnO crystals is developed based on the co‐precipitation method. ZnO crystals at different morphologies, including needle‐like, rod‐like, flower‐like, spindle‐like, and nanospheres, are successfully prepared at low temperature. The influence of reaction temperature, pH value, ZnCl2 feeding concentration, and reaction time on the morphology, size, and purity of ZnO crystals is systematically investigated. The results of X‐ray diffraction show that the high‐purity ZnO can only be produced when the reaction temperature is above 75 °C. The band gap energies of ZnO evaluated by UV–vis diffuse reflectance spectroscopy increase with the synthesis temperature. According to scanning electron microscope (SEM) findings, the size of ZnO crystals is regulated by controlling the reaction time. Interestingly, the morphology of ZnO crystals is changed from 1D to 3D structure by controlling the feeding concentration of ZnCl2. Moreover, a concentration difference mechanism of ZnO crystal morphology evolution is proposed by monitoring the real‐time concentration of free Zn2+ ions.

Ratiometric Bioluminescent Zinc Sensor Proteins to Quantify Serum and Intracellular Free Zn2.

Fluorescent Zn2+ sensors play a pivotal role in zinc biology, but their application in complex media such as blood serum or plate reader-based cellular assays is hampered by autofluorescence and light scattering. Bioluminescent sensor proteins provide an attractive alternative to fluorescent sensors for these applications, but the only bioluminescent sensor protein developed so far, BLZinCh, has a limited sensor response and a suboptimal Zn2+ affinity. In this work, we expanded the toolbox of bioluminescent Zn2+ sensors by developing two new sensor families that show a large change in the emission ratio and cover a range of physiologically relevant Zn2+ affinities. The LuZi platform relies on competitive complementation of split NanoLuc luciferase and displays a robust, 2-fold change in red-to-blue emission, allowing quantification of free Zn2+ between 2 pM and 1 nM. The second platform was developed by replacing the long flexible GGS linker in the original BLZinCh sensor by rigid polyproline linkers, yielding a series of BLZinCh-Pro sensors with a 3–4-fold improved ratiometric response and physiologically relevant Zn2+ affinities between 0.5 and 1 nM. Both the LuZi and BLZinCh-Pro sensors allowed the direct determination of low nM concentrations of free Zn2+ in serum, providing an attractive alternative to more laborious and/or indirect approaches to measure serum zinc levels. Furthermore, the genetic encoding of the BLZinCh-Pro sensors allowed their use as intracellular sensors, where the sensor occupancy of 40–50% makes them ideally suited to monitor both increases and decreases in intracellular free Zn2+ concentration in simple, plate reader-based measurements, without the need for fluorescence microscopy.

One-dimensional ZnO micro/nanostructures: deep insight into the growth mechanism and fine control of the microscopic morphology.

One-dimensional zinc oxide arrays with various finely controlled microscopic morphologies are grown on fluorine-doped tin oxide substrates by a hydrothermal method. The relationship between the microscopic morphologies and the reaction conditions are studied deeply. It is found that although all the studied reaction parameters, like reaction time, pH value, concentration of the reactants and so on, can affect the microstructures of the resultant products, what they affect, in essence, is the concentration of free Zn2+ ions of the solution. By exploring the evolution of the microstructure of the one-dimensional zinc oxide crystals, it is proved that the formation of the various microscopic morphologies is a result of the competition between the kinetic control and the thermodynamic control during the crystal growth, which in turn is mainly determined by the concentration of the free Zn2+ ions in the solution. The in-depth exploration of the growth mechanism of zinc oxide and the fine control of its microscopic morphologies is expected to provide advanced materials for current and future cutting-edge applications of zinc oxide. It is also expected that the growth mechanism reported here can provide theoretical support for achieving other oxide materials whose microstructure can be finely controlled.

Distinct pathways for zinc metabolism in the terrestrial slug Arion vulgaris

In most organisms, the concentration of free Zn2+ is controlled by metallothioneins (MTs). In contrast, no significant proportions of Zn2+ are bound to MTs in the slug, Arion vulgaris. Instead, this species possesses cytoplasmic low-molecular-weight Zn2+ (LMW Zn) binding compound that divert these metal ions into pathways uncoupled from MT metabolism. Zn2+ is accumulated in the midgut gland calcium cells of Arion vulgaris, where they associate with a low-molecular-weight ligand with an apparent molecular mass of ~ 2,000 Da. Mass spectrometry of the semi-purified LMW Zn binding compound combining an electrospray ion source with a differential mobility analyser coupled to a time-of-flight mass spectrometer revealed the presence of four Zn2+-containing ion signals, which arise from disintegration of one higher MW complex resulting in an ion-mobility diameter of 1.62 nm and a molecular mass of 837 Da. We expect that the novel Zn2+ ion storage pathway may be shared by many other gastropods, and particularly species that possess Cd-selective MT isoforms or variants with only very low affinity to Zn2+.

Some Plant Enzymes Are Highly Sensitive to Inhibition by Zinc Ions

It is well known that excessive zinc accumulation is harmful to plant cells. Though, it is uncertain at which levels of intracellular availability zinc begins to exert its deleterious action on cell physiology, as the current estimations of zinc in vitro acting concentrations seem to be non-physiologically high (micro- or millimolar). Total esterase activity in plant cells is sensitive to different adverse factors and is often used to access cell physiological activity. Therefore, we determined the effect of different zinc availability levels in vitro on the total esterase activity in extracts from rapeseed (Brassica napus L.) roots and leaves. We founded that esterase activity was drastically decreased by different protein-affecting factors, namely chaotropic salts, detergents, elevated temperatures and denaturing agents. The utilization of reaction media with exactly specified concentrations of free Zn2+ ions demonstrated that total esterase activity is substantially reduced already at tens of nM Zn2+, which is several orders of magnitude lower than was described earlier. Total phosphatase activity in extracts was even more sensitive, being drastically reduced at free Zn2+ concentrations of few nM. Therefore, the plant cellular processes can be adversely affected at very low free Zn2+ concentrations.

Challenge to assess the toxic contribution of metal cation released from nanomaterials for nanotoxicology – the case of ZnO nanoparticles

The identification of physicochemical factors that govern toxic effects of nanomaterials (NMs) is important for the safe design and synthesis of NMs. The release of metal cations from NMs in cell culture medium and the role of the metal cations in cytotoxicity are still under dispute. Here, we report that removal of NMs such as ZnO nanoparticles (NPs) by centrifugation, the procedure commonly used for the estimation of released ion concentration in nanotoxicology, was incomplete even at a relative centrifugal force of 150,000 × g. In this sense, the Zn concentration in supernatant measured by inductively coupled plasma-mass spectrometry cannot be regarded as the concentration of free Zn(2+) ions which were released from ZnO NPs in cell culture medium. This suggests the urgent need to develop relevant analytical techniques for nanotoxicology. The toxic contribution of released Zn(2+) ions to the A549 cell lines was estimated to be only about 10%. We conclude that the cytotoxicity associated with ZnO NPs is not a function of the Zn concentration, suggesting that other factors play an important role in the toxic effect of ZnO NPs.

Measuring steady-state and dynamic endoplasmic reticulum and Golgi Zn 2+ with genetically encoded sensors

Zn(2+) plays essential roles in biology, and cells have adopted exquisite mechanisms for regulating steady-state Zn(2+) levels. Although much is known about total Zn(2+) in cells, very little is known about its subcellular distribution. Yet defining the location of Zn(2+) and how it changes with signaling events is essential for elucidating how cells regulate this essential ion. Here we create fluorescent sensors genetically targeted to the endoplasmic reticulum (ER) and Golgi to monitor steady-state Zn(2+) levels as well as flux of Zn(2+) into and out of these organelles. These studies reveal that ER and Golgi contain a concentration of free Zn(2+) that is 100 times lower than the cytosol. Both organelles take up Zn(2+) when cytosolic levels are elevated, suggesting that the ER and Golgi can sequester elevated cytosolic Zn(2+) and thus have the potential to play a role in influencing Zn(2+) toxicity. ER Zn(2+) homeostasis is perturbed by small molecule antagonists of Ca(2+) homeostasis and ER Zn(2+) is released upon elevation of cytosolic Ca(2+) pointing to potential exchange of these two ions across the ER. This study provides direct evidence that Ca(2+) signaling can influence Zn(2+) homeostasis and vice versa, that Zn(2+) dynamics may modulate Ca(2+) signaling.

Zn2+-Dependent Misfolding of the p53 DNA Binding Domain

The DNA binding domain (DBD) of p53 folds by a complex mechanism that involves parallel pathways and multiple intermediates, both on- and off-pathway. This heterogeneity renders DBD particularly susceptible to misfolding and aggregation. The origins of parallel folding mechanisms are not well understood. DBD folding heterogeneity may be caused by the presence of the single bound Zn2+. To test that hypothesis, we carried out kinetic folding studies of DBD in its Zn2+-free form (apoDBD) and in the presence of various concentrations of free Zn2+ and the Zn2+-nitrilotriacetate (NTA) complex. Folding kinetics of apoDBD and DBD are similar, although apoDBD folds faster than DBD at some urea concentrations. The principle consequence of Zn2+ removal is to accelerate unfolding and simplify it from two exponential phases to one. Metal binding interactions are therefore not responsible for the observed complexity of the folding reaction. A slight stoichiometric excess of free Zn2+ arrests folding and traps the protein in a misfolded state in which Zn2+ is bound to nonphysiological ligands. Folding can be rescued by providing metal ions in the form of the NTA-Zn2+ complex, which simultaneously protects against misligation and provides a source of Zn2+ for regenerating the functional protein. This chemical metallochaperone strategy may be an effective means for improving folding efficiency of other metal binding proteins. The findings suggest that, in vivo, DBD must fold in an environment where free Zn2+ concentration is low and its bioavailability is carefully regulated by cellular metallochaperones.

The chemical speciation of zinc in human saliva: possible correlation with reduction of the symptoms of the common cold produced by zinc gluconatecontaining lozenges

This work attempts to correlate the effectiveness of treatment with zinc gluconate lozenges in reducing the symptoms of common colds as measured in seven double-blind clinical trials in 648 volunteers, with (1) the total amount of zinc gluconate administered and (2) the salivary concentration of specific zinc species. The speciation was assessed using the JESS computer simulation of speciation in our saliva model. The results show that there is a significant correlation between the biological response and the concentration of free Zn2+aq ions in saliva at pH 5.5. (P≅0.01) or pH 6.75 (P<0.05), whereas there is no obvious correlation between biological response and the total amount of zinc present following the dissolution of a zinc gluconate lozenge in the mouth. The results suggest that optimal activity requires salivary freeZn2+aq ion concentrations of >4 and >8 mmol dm−3 for saliva at pH = 6.75 and pH = 5.5, respectively.

Production of an expression system for a synaptobrevin fragment to monitor cleavage by botulinum neurotoxin B.

Recombinant DNA techniques were used to develop an expression system for a 51-amino acid peptide fragment that encompasses residues 44-94 of human synaptobrevin 2. This protein is associated with secretory vesicles of nerve terminals and is a substrate for four of the seven serotypes of botulinum neurotoxin (BoNT). The DNA for the recombinant peptide was amplified by the polymerase chain reaction and cloned into the pTrxFus vector. The resulting synaptobrevin peptide was expressed as a thioredoxin fusion protein in E. coli and released into the medium by osmotic lysis. The 18.7-kDa thioredoxin-synaptobrevin protein, designated as TSB-51, is intended for use in a cell-free assay to test potential inhibitors of BoNT/B-mediated proteolysis of synaptobrevin with the ultimate aim of developing clinically effective therapeutic agents to counteract botulism. Incubation of TSB-51 with the purified light chain of BoNT/B resulted in proteolysis which was evident within 30 min and increased with time until completion (approximately 4 hr). Cleavage of TSB-51 appeared to be at the appropriate BoNT/B cleavage site as indicated by a reduced intensity of the 18.7-kDa band and the appearance of a band at 16.4 kDa on Tris-tricene polyacrylamide gradient gels. The concentration of free Zn2+ had a significant effect on the cleavage rate; low Zn2+ concentrations stimulated substrate cleavage, whereas high concentrations were inhibitory. Cleavage was not significantly depressed by the naturally occurring metalloprotease inhibitor phosphoramidon when tested at concentrations up to 5 mM. TSB-51 appears to be a useful substrate for studying BoNT/B and is expected to aid in the discovery of effective BoNT inhibitors.

The concentrations of free Mg2+ and free Zn2+ in equine blood plasma.

The enzyme phosphoglucomutase can be used as a metal ion indicator to measure the concentrations of free Mg2+ and free Zn2+ in physiological fluids. In horse plasma, the concentration of free Mg2+ is close to 0.5 mM, whereas that of free Zn2+ is about 2 X 10(-10) M, although numerous physiological roles for Zn2+ have been postulated that would require free Zn2+ concentration orders of magnitude higher than this. A titration of plasma with Zn2+ shows that the fractional increase in free Zn2+ is essentially the same as the fractional increase in total exchangeable Zn2+, and the results are consistent with a model in which essentially all of the Zn2+ in plasma is bound to albumin. Regardless of the model, the buffering capacity of plasma for free Zn2+ is intrinsically low; however, its capacity relative to the total (exchangeable) Zn2+ present is maximal. The implications of this type of buffering for homeostasis of plasma Zn2+ are considered. Treatment of plasma with a strong reducing agent such as dithiothreitol (0.1 mM) substantially increases the apparent binding of Zn2+ and thus reduces the free Zn2+ concentration. However, the concentration of free Zn2+ appears to be insensitive to decreases in the physiological concentrations of reduced glutathione and cysteine. The concentrations of free Zn2+ and free Mg2+ in plasma are similar to those that have been reported for muscle tissue (rabbit). Their ratio is about 4 X 10(-7). The physiological implications of these concentrations are considered. In some cases, if the Zn2+ and Mg2+ complexes of an uncharacterized vertebrate protein exhibit significantly different properties, their relative importance under physiological conditions can be approximated by evaluating those of the mixed complexes present in a solution that contains the physiological concentration of free Mg2+, plus Zn2+ buffered with histidine, at the appropriate pH and ionic strength. Other metal ion/chelon systems that come close to reproducing the concentrations of free Mg2+ and free Zn2+ in horse plasma also are considered.

The oxygen-linked zinc-binding site of human haemoglobin.

Zn2+ is known to increase the 02 affinity of human haemoglobin. Previous data suggested that Zn2+ exerts its effect by directly binding to haemoglobin, rather than by competing with or binding to 2,3-bisphosphoglycerate. It was also shown that there are two 02-linked zinc-binding sites in haemoglobin, and that Zn2+ does not significantly alter haemoglobin co-operativity or the alkaline Bohr effect. The effect of Zn2+ on 02 affinity of haemoglobin can also be observed for other haemoglobins as diverse as those of cow and chicken. This paper presents new data on the haemoglobin-zinc interaction for normal haemoglobin, des-His146beta-haemoglobin and N-ethylsuccinimide-haemoglobin of humans. For normal haemoglobin (0.05 mM in tetramers), at 20 degrees C in buffer containing 0.1 M-Cl-, 02-dissociation-curve experiments showed that the addition of 0.4-0.5 mM-ZnS04 did not change the Bohr effect between pH 6.71 and 7.29. Similar experiments, with "zinc-ion buffers", showed that the value of the Hill coefficient, h, decreased only slightly if the concentration of free Zn2+ was held constant. For N-ethylsuccinimide-haemoglobin, Zn2+ caused less increase in O2 affinity than for normal haemoglobin. These studies, together with data on the equilibrium binding of Zn2+ to oxy-, deoxy- and des-His146beta-haemoglobins, suggest that zinc is chelated in oxyhaemoglobin by at least three amino acids, two of which are histidine-146beta and cysteine-93beta.