Electrochemical survey of the chain length influence in phytochelatins competitive binding by cadmium

Multivariate curve resolution with alternating least squares (MCR-ALS) was applied to voltammetric data obtained in the analysis of the competitive binding of glutathione (GSH) and phytochelatins [(γGlu-Cys)_n-Gly, PC_n, n = 2-5] by Cd²⁺. The displacements between ligands and chain length influence on the competitive binding of PC_n toward Cd²⁺ were investigated. The analysis of the resulting pure voltammograms and concentration profiles of the resolved components suggests that ligands containing more thiol groups are able to displace the shortest chain ligands from their metal complexes, whereas the opposite does not happen. However, when the length of the chain surpasses that of PC₃, the binding capacity of the molecule still increases (i.e., it can bind more metal ions), but the position and shape of the voltammetric signals practically rest unchanged. This suggests that at this level, the stability of metal binding could depend more on the nature of the binding sites separately than on the quantity of the sites (i.e., the chain length).     

Cadmium(II)-stimulated enzyme activation of Arabidopsis thaliana phytochelatin synthase 1

Phytochelatin (PC), a class of heavy metal-binding peptides, is synthesized from the tripeptide glutathione (GSH) and/or previously synthesized PC in a reaction mediated by PC synthase (PCS). In the present study, the PC production rate catalyzed by recombinant Arabidopsis PCS1 (rAtPCS1) in the presence of a constant free Cd(II) level increased steadily and the kinetic parameters were approximated using a substituted-enzyme mechanism in which GSH and bis(glutathionato)cadmium acted as co-substrates. In contrast, the PC production rate as a function of GSH concentration at a constant total Cd(II) concentration reached a maximum, which shifted toward higher GSH concentrations as the concentration of Cd(II) was increased. These observations are consistent with the suggestion that rAtPCS1 possesses a Cd(II) binding site where Cd(II) binds to activate the enzyme. The affinity constant, optimized using a one-site mathematical model, successfully simulated the experimental data for the assay system using lower concentrations of Cd(II) (5 or 10 μM) but not for the assay using higher concentrations (50 or 500 μM), where a sigmoidal increase in PCS activity was evident. Furthermore, the PCS activity determined at a constant GSH concentration as a function of Cd(II) concentration also reached a maximum. These findings demonstrate that rAtPCS1 also possesses a second Cd(II) binding site where Cd(II) binds to induce an inhibitory effect. A two-site mathematical model was applied successfully to account for the observed phenomena, supporting the suggestion that rAtPCS1 possesses two Cd(II) binding sites.     

A new and highly sensitive fluorescence assay for collagenase-like peptidase activity.

A highly sensitive fluorescence assay for collagenase-like peptidase (CL-peptidase) has been developed using a newly synthesized substrate, (succinyl-Gly-Pro-Leu-Gly-Pro)-4-methylcoumaryl-7-amide (Suc-GPLGP-MCA). Suc-GPLGP-MCA was hydrolyzed at the Leu-Gly bond by CL-peptidase, (Gly-Pro)-4-methylcoumaryl-7-amide liberated by the enzyme was immediately hydrolyzed to Gly-Pro and 7-amino-4-methylcoumarin (AMC) by an excess of an auxiliary enzyme, X-prolyl dipeptidyl-aminopeptidase, and the fluorescence intensity of the AMC was measured at 460 nm with excitation at 380 nm. When assayed by this method, CL-peptidase partially purified from chick embryo showed a pH optimum at 8.0 and a K_m value of 4.0 × 10^?4m toward Suc-GPLGP-MCA. Under the optimum condition, the reaction proceeded linearly up to 4 h. The CL-peptidase activity was found in normal human sera by this method and the mean and standard deviation of the activity was 0.59 ± 0.10 nmol/min/ml of serum (n = 10). This assay was also applicable for the CL-peptidase in human liver and kidney. The results suggest that the CL-peptidase assayed by this new substrate may be different from the “PZ-peptidase” which cleaves a synthetic substrate for collagenase-like peptidase, 4-phenylazobenzyloxycarbonyl (PZ)-Pro-Leu-Gly-Pro-d-Arg (PZ-peptide). The new peptide, Suc-GPLGP-MCA, was found not to be a substrate for specific collagenase from tadpole.     

Heavy metal detoxification in higher plants - a review

A set of heavy-metal-complexing peptides was isolated from plants and plant suspension cultures. The structure of these peptides was established as (γ-glutamic acid-cysteine)_n-glycine (n=2–11) [(γ-Glu-Cys)_n-Gly]. These peptides appear upon induction of plants with metals of the transition and main groups (Ib-Va, Z=29−83) of the periodic table of elements. These peptides, called phytochelatins (PC), are induced in all autotrophic plants so far analyzed, as well as in select fungi. Some species of the order Fabales and the family Poaceae synthesize aberrant PC that contain, at their C-terminal end, either β-alanine, serine or glutamic acid. For this group of peptides the name iso-PC is proposed. The biosynthesis of PC proceeds by metal activation of a constitutive enzyme that uses glutathione (GSH) as a substrate; this enzyme is a γ-glutamylcysteine dipeptidyl transpeptidase which was given the trivial name PC synthase. It catalyzes the following reaction: γ-Glu-Cys-Gly+(γ-Glu-Cys)_n-Gly→(γ-Glu-Cys)_n+1-Gly+Gly. The plant vacuole is the transient storage compartment for these peptides. They probably dissociate, and the metal-free peptide is subsequently degraded. Sequestration of heavy metals by PC confers protection for heavy-metal-sensitive enzymes. The isolation of a Cd²⁺-sensitive cadl mutant of Arabidopsis thaliana, that is deficient in PC synthase, demonstrates conclusively the importance of PC for heavy metal tolerance. In spite of the fact that nucleic acid sequences and proteins are found in higher plants that have distant homology to animal metallothioneins, there is absolutely no experimental evidence that these ‘plant metallothioneins’ are involved in the detoxification of heavy metals. PC synthase will be an interesting target for biotechnological modification of heavy metal tolerance in higher plants.     

Assay for human erythrocyte pyridoxine kinase

An accurate and rapid method for the assay of pyridoxine kinase in human erythrocytes has been developed. The procedure involves the separation of the radioactive product from the substrate with Dowex 50 resin in a test tube. Using the assay designed, we found that human red blood cells have a pyridoxine kinase activity of 1.381 nmole/min/g of hemoglobin (n = 25, SE = 0.051), and the enzyme has a K_m of approximately 1.72 × 10^?6m for pyridoxine. Pyridoxine phosphate was identified as the main product of the assay reaction catalyzed by human erythrocyte pyridoxine kinase in crude hemolysates.     

Stereospecific ligands and their complexes. V. Synthesis, characterization and antimicrobial activity of palladium(II) complexes with some alkyl esters of (S,S)-ethylenediamine-N,N′-di-2-propanoic acid

Three new ligands and their palladium(II) complexes of general formula [PdCl₂(R₂-S,S-eddp)] (R = n-propyl, n-butyl and n-pentyl) have been synthesized and characterized by microanalysis, infrared and ¹H and ¹³C NMR spectroscopy. Antimicrobial activity of these ligands and complexes was tested by microdilution method and both minimal inhibitory and microbicidal concentration were determined. These tested complexes demonstrated the significant antifungal activity against pathogenic fungi Aspergillus flavus and Aspergillus fumigatus. On the other hand, these complexes demonstrated moderate antibacterial activity.     

Phytochelatin synthesis in response to elevated CO2 under cadmium stress in Lolium perenne L

The increasing atmospheric CO₂ and heavy metal contamination in soil are two of the major environmental problems. Knowledge of the Cd stress coping mechanisms is needed to understand the regulation of the plants’ metabolism under the increasing atmospheric CO₂ levels. Lolium perenne L. was grown hydroponically under two concentrations of atmospheric CO₂ (360 and 1000 μL L⁻¹) and six concentrations of cadmium (0-160 μmol L⁻¹) to investigate Cd uptake, Cd transportation, and variations in phytochelatin (PC) concentration. Cd concentrations in roots and shoots were decreased, but transport index (Ti) was increased under elevated CO₂ compared to ambient CO₂. Regardless of CO₂ concentrations, Cd and PC concentrations, especially the concentrations of high molecular weight PCs (PC₄, PC₅, PC₆) were higher with increasing Cd concentration in growth media and longer Cd exposure time. Under the elevated CO₂, more high molecular weight PCs (PC₄, PC₅, PC₆) in shoots and roots were synthesized compared to ambient CO₂, with higher SH:Cd ratio in roots as well. These results indicate that under elevated CO₂, L. perenne may be better protected against Cd stress with higher biomass, lower Cd concentration and better detoxification by phytochelatins.     

Biochemical and structural properties of gamma-glutamyl transpeptidase from Geobacillus thermodenitrificans: An enzyme specialized in hydrolase activity

Gamma-glutamyltranspeptidases (γ-GTs) catalyze the transfer of the gamma-glutamyl moiety of glutathione and related gamma-glutamyl amides to water (hydrolysis) or to amino acids and peptides (transpeptidation) and play a key role in glutathione metabolism. Recently, γ-GTs have been considered attractive pharmaceutical targets for cancer and useful tools to produce γ-glutamyl compounds. To find out γ-GTs with special properties we have chosen microorganisms belonging to Geobacillus species which are source of several thermostable enzymes of potential interest for biotechnology. γ-GT from Geobacillus thermodenitrificans (GthGT) was cloned, expressed in Escherichia coli, purified to homogeneity and characterized. The enzyme, synthesized as a precursor homotetrameric protein of 61-kDa per subunit, undergoes an internal post-translational cleavage of the 61 kDa monomer into 40- and 21-kDa shorter subunits, which are then assembled into an active heterotetramer composed of two 40- and two 21-kDa subunits. The kinetic characterization of the hydrolysis reaction using l-glutamic acid γ-(4-nitroanilide) as the substrate reveals that the active enzyme has K_m 7.6 μM and V_max 0.36 μmol min/mg. The optimum pH and temperature for the hydrolysis activity are 7.8 and 52 °C, respectively. GthGT hydrolyses the physiological antioxidant glutathione, suggesting an involvement of the enzyme in the cellular defense mechanism against oxidative stress. Unlike other γ-GTs, the mutation of the highly conserved catalytic nucleophile, Thr353, abolishes the post-translational cleavage of the pro-enzyme, but does not completely block the hydrolytic action. Furthermore, GthGT does not show any transpeptidase activity, suggesting that the enzyme is a specialized γ-glutamyl hydrolase. The GthGT homology-model structure reveals peculiar structural features, which should be responsible for the different functional properties of the enzyme and suggests the structural bases of protein thermostability.     

A continuous coupled polarographic assay for trehalase.

A continuous, coupled polarographic assay, which couples trehalose hydrolysis to O₂ consumption using glucose oxidase (EC 1.1.3.4) and catalase (EC 1.11.1.6) as ancillary enzymes has been developed for the measurement of trehalase (α-α′-trehalose 1-d-glucohydrolase, EC 3.2.1.28) activity. With this procedure, O₂ consumption was a linear function of time and the coupled reaction rate was directly proportional to the amount of protein assayed with both crude and partially purified enzyme preparations. The limits of sensitivity with this assay correspond to the production of 2.5 nmol of glucose/min. The validity of this assay was confirmed by comparative studies with a discontinuous colorimetric assay for the quantitation of glucose. In addition, the applicability of this assay was appraised by determining the K_m of the enzyme for trehalose. The value obtained with the polarographic assay (i.e., 1.3 ± 0.1 mm trehalose) showed excellent agreement with that obtained using a discontinuous colorimetric method (i.e., 1.2 mm trehalose). Thus the equivalence and applicability studies with the polarographic assay demonstrated that this procedure is a valid and sensitive method for the rapid quantitation of trehalase activity.     

Phytochelatin–metal(loid) transport into vacuoles shows different substrate preferences in barley and Arabidopsis

Cadmium (Cd) and arsenic (As) are toxic to all living organisms, including plants and humans. In plants, Cd and As are detoxified by phytochelatins (PCs) and metal(loid)‐chelating peptides and by sequestering PC–metal(loid) complexes in vacuoles. Consistent differences have been observed between As and Cd detoxification. Whereas chelation of Cd by PCs is largely sufficient to detoxify Cd, As–PC complexes must be sequestered into vacuoles to be fully detoxified. It is not clear whether this difference in detoxification pathways is ubiquitous among plants or varies across species. Here, we have conducted a PC transport study using vacuoles isolated from Arabidopsis and barley. Arabidopsis vacuoles accumulated low levels of PC₂–Cd, and vesicles from yeast cells expressing either AtABCC1 or AtABCC2 exhibited negligible PC₂–Cd transport activity compared with PC₂–As. In contrast, barley vacuoles readily accumulated comparable levels of PC₂–Cd and PC₂–As. PC transport in barley vacuoles was inhibited by vanadate, but not by ammonium, suggesting the involvement of ABC‐type transporters. Interestingly, barley vacuoles exhibited enhanced PC₂ transport activity when essential metal ions, such as Zn(II), Cu(II) and Mn(II), were added to the transport assay, suggesting that PCs might contribute to the homeostasis of essential metals and detoxification of non‐essential toxic metal(loid)s.     

Spectrophotometric assay for protease activity in ionic liquids using chromogenic substrates

A new spectrophotometric assay has been developed to evaluate protease activity in ionic liquids (ILs). The assay consists of two strategies to enable real-time spectrometric analysis of enzymatic reaction in ILs. First, enzymes are modified with a comb-shaped poly(ethylene glycol), PM₁₃, to obtain a transparent enzyme solution in IL. Second, a chromogenic substrate is used to follow the enzymatic reaction in IL. p-Nitroaniline-derivatized substrates are subjected to protease-catalyzed alcoholysis to release chromogenic p-nitroaniline that can be quantitatively detected by a UV-Vis spectrophotometer. By using this method, we can evaluate protease activity in ILs quite easily without separation of products from the reaction mixture. The availability of the novel assay system was demonstrated in a kinetic analysis of subtilisin-catalyzed reaction in the IL 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide ([Emim][Tf₂N]) under different reaction conditions. Because two different serine proteases, subtilisin and α-chymotrypsin, substantially retained its original substrate specificity in the IL, the assay can be extended to other enzymes by using suitable chromogenic substrates.     

Three families of thiol peptides are induced by cadmium in maize

Phytochelatins ((γGluCys)_nGly) are synthesized from glutathione by plants exposed to metals like Cd²⁺, Cu²⁺ and Zn²⁺. An intracellular complex formed by phytochelatins with Cd²⁺ and sulfide is thought to detoxify the metal possibly by sequestration in the vacuole. It was found that maize seedlings exposed to Cd²⁺ produced phytochelatins and two additional families of cysteina-containing peptides, (γGluCys)_n and (γGluCys)_nGlu. All thiol peptides with n = 2 and 3 were purified and their structure characterized by tandem mass spectrometry. For maize plants exposed to Cd²⁺ for 7 days, phytochelatins were synthesized preferentially in the first 24 h whereas the amounts of (γGluCys)_n and (γGluCys)_nGlu were the highest thereafter. This was probably due to an initial large pool of glutathione available in control plants compared with a dearth of γGluCys and no detectable γGluCysGlu. The (γGluCys)_nGlu peptides were induced exclusively by Cd²⁺ as they were below the detection limit in control seedlings that contained low amounts of phytochelatins and (γGluCys)_n. Since the Cys moiety of the peptides is essential for binding Cd²⁺, a role for accumulated (γGluCys)_n and (γGluCys)_nGlu in detoxifying Cd²⁺ in plants must be considered.     

A radiotracer enzyme assay for phosphofructokinase using ( , , -32P) adenosine triphosphate

A radiotracer enzyme assay for phosphofructokinase using adenosine 5′-triphosphate[α,β,γ-³²P] is described in this paper. Here the rates of appearance of both [1-³²P]d-fructose 1,6-diphosphate and [α,β-³²P] adenosine 5′-diphosphate were followed to establish enzyme activity. The unique advantages of multiple rate determinations in a single reaction sequence which accrue from the use of a readily available multiply labeled cosubstrate are discussed. By an extension of this approach other labeled(¹) nucleotides of the type, N(¹P)_n, and enzymes in the Enzyme Commision categories, EC 2.7(phosphotransferases) and EC 6.1–6.4(ligases) are equally amenable to radionuclide assay.     

Studies on the synthesis of short-chain geranyl esters catalysed by Fusarium oxysporum esterase in organic solvents

A novel esterase isolated from Fusarium oxysporum was investigated for the synthesis of short-chain esters of geraniol by alcoholysis and direct esterification reactions in organic solvents. The enzyme was used as a dried powder (i.e., not immobilized). The reaction parameters affecting the enzyme behavior such as the nature of organic solvent and acyl donor, the concentration of substrates and the water activity of the system were studied. High yields (80–90%) were obtained by both approaches (alcoholysis and direct esterification) at low values of water activity (a_w=0.11) in n-hexane. The enzyme retain its catalytic activity even after fifth reuse in n-hexane at a_w=0.11, demonstrating its stability and efficiency under the conditions of this study.     

The use of Fourier transform infrared spectroscopy to assay for urease from Pseudomonas aeruginosa and Canavalia ensiformis

A novel assay method was investigated for urease (EC 3.5.1.5) from Pseudomonas aeruginosa and Canavalia ensiformis by Fourier transform infrared spectroscopy. This enzyme catalyzed the hydrolysis of urea in phosphate buffer in deuterium oxide (²H₂O). The intensities of the bicarbonate bands maxima at 1625 and 1365 cm⁻¹ and of the amide I band at 1605 cm⁻¹ were measured as a function of time to study the kinetics of urea hydrolysis. The extinction coefficients ε of urea and bicarbonate were determined to be 0.72, 0.48, and 0.56 mM⁻¹ cm⁻¹ at 1625, 1605, and 1365 cm⁻¹, respectively. The initial velocity is proportional to the enzyme concentration by using the ureases from both C.ensiformis and P. aeruginosa. The kinetic constants (V_max, K_m, and K_cat) determined by Lineweaver-Burk plot were 532.2  U mg⁻¹ protein, 6.4 mM, and 806.36 s⁻¹, respectively. These data are in agreement with the results obtained by a spectrophotometric method using a linked assay based on glutamate dehydrogenase in aqueous media. Therefore, this spectroscopic method is highly suited to assay for urease activity and its kinetic parameters by using either cell-free extracts or purified enzyme preparations with an additional advantage of performing a real-time measurement of urease activity.     

Silica supported 12-tungstophosphoric acid catalysts for synthesis of 1,4-dihydropyridines under solvent-free conditions

12-Tungstophosphoric acid (PW) supported on different metal oxides (SiO₂, γ-Al₂O₃, KSF, K10) and activated carbon were prepared by impregnation method and their catalytic performances were evaluated in three component condensation of benzaldehyde, ethyl acetoacetate and ammonium acetate to afford corresponding 1,4-dihydropyridine. A high catalytic activity was found over silica supported PW. Effect of PW loading, catalyst loading and solvent was studied to introduce the best reaction condition. Based on the above experimental finding, catalytic performances was optimized with a loading of 40% PW onto SiO₂ (0.2 g) under solvent-free condition. The characterization data derived from FT-IR, XRD, and TGA-DSC techniques reveal that the PW on silica support exists in Keggin structure. In addition, acidity measurements were performed by potentiometric titration with n-butylamine. The activity of the catalysts is strongly dependent on their acidic characteristic which, in turn, depended on PW loading. Finally, a series of 4-aryl, N-alkyl, and N-aryl substituted 1,4-dihydropyridines have been synthesized in high to excellent yield in short reaction times. PW/SiO₂ was found to be reusable and a considerable catalytic activity still could be achieved after fourth run.     

Development of surface‐engineered yeast cells displaying phytochelatin synthase and their application to cadmium biosensors by the combined use of pyrene‐excimer fluorescence

The development of simple, portable, inexpensive, and rapid analytical methods for detecting and monitoring toxic heavy metals are important for the safety and security of humans and their environment. Herein, we describe the application of phytochelatin (PC) synthase, which plays a critical role in heavy metal responses in higher plants and green algae, in a novel fluorescent sensing platform for cadmium (Cd). We first created surface‐engineered yeast cells on which the PC synthase from Arabidopsis (AtPCS1) was displayed with retention of enzymatic activity. The general concept for the sensor is based on the Cd level‐dependent synthesis of PC₂ from glutathiones by AtPCS1‐displaying yeast cells, followed by simple discriminative detection of PC₂ via sensing of excimer fluorescence of thiol‐labeling pyrene probes. The intensity of excimer fluorescence increased in the presence of Cd up to 1.0 μM in an approximately dose‐dependent manner. This novel biosensor achieved a detection limit of as low as 0.2 μM (22.5 μg/L) for Cd. Although its use may be limited by the fact that Cu and Pb can induce cross‐reaction, the proposed simple biosensor holds promise as a method useful for cost‐effective screening of Cd contamination in environmental and food samples. The AtPCS1‐displaying yeast cells also might be attractive tools for dissection of the catalytic mechanisms of PCS. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1197–1202, 2013     

A continuous coupled spectrophotometric assay for debranching enzyme activity using reducing end-specific α-glucosidase

A novel continuous spectrophotometric assay to measure the activity of the debranching enzyme and α-amylase has been developed. The assay mixture comprises the debranching enzyme (GlgX from Escherichia coli) or α-amylase (PPA from porcine pancreas), a reducing end-specific α-glucosidase (MalZ), maltodextrin-branched β-cyclodextrin (Glc_n-β-CD) as the substrate, and the glucose oxidase/peroxidase system (GOPOD). Due to its high reducing end specificity, the branch chains of the substrates are not hydrolyzed by MalZ. After hydrolysis by GlgX or PPA, the released maltodextrins are immediately hydrolyzed into glucose from the reducing end by MalZ, whose concentration is continuously measured by GOPOD at 510 nm in a thermostat spectrophotometer. The kinetic constants determined for GlgX (K_m = 0.66 ± 0.02 mM and k_cat = 76.7 ± 1.5 s⁻¹) are within a reasonable range compared with those measured using high-performance anion-exchange chromatography (HPAEC). The assay procedure is convenient and sensitive, and it requires lower concentrations of enzymes and substrate compared with dinitrosalicylic acid (DNS) and HPAEC analysis.     

Colorimetric estimation of human glucose level using γ-Fe2O3 nanoparticles: An easily recoverable effective mimic peroxidase

This article reports simple, green and efficient synthesis of γ-Fe₂O₃ nanoparticles (NPs) (maghemite) through single-source precursor approach for colorimetric estimation of human glucose level. The γ-Fe₂O₃ NPs, having cubic morphology with an average particle size of 30 nm, exhibited effective peroxidase-like activity through the catalytic oxidation of peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H₂O₂ producing a blue-colored solution. On the basis of this colored-reaction, we have developed a simple, cheap, highly sensitive and selective colorimetric method for estimation of glucose using γ-Fe₂O₃/TMB/glucose–glucose oxidase (GOx) system in the linear range from 1 to 80 μM with detection limit of 0.21 μM. The proposed glucose sensor displays faster response, good stability, reproducibility and anti-interference ability. Based on this simple reaction process, human blood and urine glucose level can be monitored conveniently.     

A high-throughput colorimetric assay to characterize the enzyme kinetic and cellular activity of spermidine/spermine N-acetyltransferase 1

Spermidine/spermine N¹-acetyltransferase 1 (SSAT1) is a key enzyme that catalyzes the catabolism of polyamines. SSAT1 is a very important enzyme because it not only maintains the homeostasis of polyamines but also is involved in many physiological and pathological events. As such, a rapid assay of SSAT1 activity is valuable in drug screening and clinical diagnostics. Here, we report a novel colorimetric assay for monitoring SSAT1 activity in zebrafish (zSSAT1). In comparison with the available SSAT1 assays, this new method is cost-effective and simple. The optimal zSSAT1 activity was obtained below 55 °C in a mild alkaline environment. The K_m values of zSSAT1 for spermidine and spermine are 55 and 182 μM, respectively, whereas putrescine is not a good substrate for zSSAT1. In addition to enzyme kinetic studies, the colorimetric assay was also used to detect the cellular activity of SSAT1. Thus, the current method is a reliable assay for determining SSAT1 activity with many potential applications in medical biology.