Assay for thiols based on reactivation of papain.
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Animals↗
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Studies on the purification of peroxidase from horseradish roots using reverse micelles.
Horseradish peroxidase (HRP) was successfully purified from horseradish roots by a two-stage reverse-micellar extraction from the dialyzed aqueous extract. The anionic surfactant AOT dissolved in isooctane was used to produce the reverse-micellar phases. The narrow pH range at which HRP solubilization occurred was exploited to remove most of the contaminant proteins in the first forward extraction. In the second extraction stage, HRP was selectively solubilized and concentrated by using a volume ratio of 10 between the aqueous and organic phases. The HRP final specific activity was 86 guaiacol U mg-1, obtained with a purification factor of 80 and yield of 46%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two overlapping bands, with HRP corresponding to that at 43.8 kDa. Image analysis on isoelectric focusing (IEF) gels showed that the HRP was 80% pure. Ion exchange liquid chromatography showed that most of the specific activity was due to the basic isoenzyme with pI 8.5, which comprises 33.5% of the product. There were high HRP losses as a precipitate at the interface when direct reverse-micellar extraction was attempted from the crude extract. It is believed that the hydrophobic environment near the haem group of the HRP basic isoenzyme favors complex formation with the surfactant, and that this is promoted at higher protein concentrations.
Calcium↗
Reduction and release of ferritin iron by plant phenolics.
The reductive release of ferritin iron by several naturally occurring o-diphenols was studied. The initial rate of iron release was quantified by spectrophotometric measurement of the Fe(ferrozine)3(2+) complex, which absorbs maximally at 562 nm. The initial rate of iron release was dependent upon o-diphenol concentration, but not on the concentration of the chromophoric chelating agent, ferrozine, Stoichiometric measurements resulted in a ratio of 2Fe(II) released per molecule of o-diphenol. The series of o-diphenols studied included, caffeic acid, chlorogenic acid, dihydrocaffeic acid, 3,4-dihydroxybenzoic acid, and several analogs. These reductants represent an oxidation reduction potential range of 0.38 volts. A direct correlation between reducing power of the o-diphenols and rate of ferritin iron release was observed. Superoxide dismutase, catalase, mannitol, or general radical traps had no effect on the rate of iron removal; however, EDTA and oxalate inhibited iron release. A mechanism for ferritin iron reduction and release by o-diphenols consistent with the experimental observations is discussed.
Animals↗
Synthesis, characterization, and antioxidative activity of vitamin B6 rare earth (III) complexes.
A series of vitamin B6 pyridoxol rare earth (III) complexes Re(PN)5Cl3. 6H2O (PN = pyridoxol; Re = La, Ce, Pr, Nd, Sm, Eu, Gd, Ho, Er, Y) were synthesized and characterized by elemental analysis, molar conductance, TGA-DTA, IR, and 1HNMR spectroscopic techniques. The antioxidative activity of complexes was determined, using the suppression ratio of activated oxygen as an indicator. The results show that the complexes have the scavenger effects for activated oxygen.
Antioxidants↗
Effects of anticoagulants upon sister-chromatid exchanges, cell-cycle kinetics, and mitotic index in human peripheral lymphocytes.
The effects of 3 blood anticoagulants, heparin, acid citrate dextrose (ACD), and ethylenediaminetetraacetic acid (EDTA) were investigated using human peripheral lymphocytes. Three different endpoints were examined: sister-chromatid exchange (SCE), cell kinetics index (CKI), and mitotic index (MI). SCEs were significantly increased in cells treated with EDTA, while the CKI and MI were significantly decreased in cultures treated with either ACD or EDTA when compared to cultures treated with heparin. These results suggest that anticoagulants may produce undesired effects upon cultured cells and indicate that the type of anticoagulant should be considered carefully prior to commencing cytogenetic studies using human peripheral lymphocytes.
Anticoagulants↗
Biochemical research on oogenesis: distribution of tRNA-linked peptides and proteins in previtellogenic oocytes of Xenopus laevis.
Peptides and proteins were detected in the deacylation products of tRNA purified from the 42S particles and from the messenger ribonucleoprotein particles (mRNPs) present in the previtellogenic oocytes of Xenopus laevis. Only a small fraction of particle tRNA carries a peptide or protein chain. The bulk of particle tRNA is simply aminoacylated. The tRNA-linked peptide chains of the particles appear to turn over more slowly in vivo than aminoacyl tRNA. These chains could arise in the particles by a peptidyl transfer reaction similar to that carried out by the ribosome.
Acylation↗
Studies on the inhibition of gluconeogenesis by oxalate.
Oxalate was shown to enter isolated rat hepatocytes and to inhibit gluconeogenesis from lactate, pyruvate, and alanine, but not from glutamine, proline, propionate or dihydroxyacetone. Oxalate apparently acts by inhibiting pyruvate carboxylase (EC 6.4.1.1.). It is known to inhibit the isolated enzyme, and inhibition of gluconeogenesis was much greater in a bicarbonate-deficient medium where pyruvate carboxylase activity limits the overall rate of the pathway. A slight inhibition of gluconeogenesis from asparagine was observed, suggesting that oxalate may also inhibit gluconeogenesis at another site. Chelation of extracellular Ca2+ does not contribute to the inhibition of gluconeogenesis. Compared to oxalate, other Ca2+ chelators have little effect upon gluconeogenesis. Also, oxalate inhibits gluconeogenesis effectively both in low Ca2+ medium and in medium containing 2.6 mM Ca2+. Chelation of intracellular Ca2+ also appears to be of little importance, since oxalate does not block the glycogenolytic effects of epinephrine, vasopressin, and angiotensin which are thought to act via Ca2+ as the second messenger. The inhibition of gluconeogenesis could conceivably contribute to the toxic actions of oxalate and to the hypoglycemic action of dichloroacetate, a compound that is metabolized to oxalate. However, oxalate did not cause hypoglycemia in the suckling rat, a model in vivo system very dependent upon gluconeogenesis for maintenance of normal blood glucose levels. Thus, inhibition of gluconeogenesis is probably of little importance in oxalate toxicity and the hypoglycemic effects of dichloroacetate.
Angiotensins↗
Glutathione reduces cytoplasmic vanadate. Mechanism and physiological implications.
The mechanism by which cells reduce cytoplasmic vanadium(V) (vanadate) to vanadium(IV) was investigated using the human red cell as a model system. Vanadate uptake by red cells occurs with a rapid phase involving chemical equilibration across the plasma membrane and a slower phase resulting in a high concentration of bound vanadium(IV). The slow phase was inhibited in glucose-starved cells and restored upon addition of glucose indicating an energy requirement for this process. The time course of vanadium(IV) appearance (monitored by EPR spectroscopy of intact cells) paralleled the slow phase of uptake indicating that this phase involves vanadium reduction. The reduction of intracellular vanadate to vanadium(IV) was nearly quantitative after 23 h. The intracellular reduction is not enzymatic, since a similar time course of vanadium reduction and binding to hemoglobin was observed when glutathione was added to a hemoglobin + vanadate solution in vitro. Vanadium(IV) binding to hemoglobin was reduced by addition of ATP, 2,3-diphosphoglycerate or EDTA, probably through chelation of the cation. The stability constant of the ATP-vanadium (IV) complex was determined to be 150 M-1 at pH 4.9. The time course of red cell vanadate uptake and reduction was followed in the concentration range in which approximately 60% inhibition of the (Na+ + K+)-ATPase is observed. It is concluded that vanadate is reduced by cytoplasmic glutathione in this concentration range and that the reduction explains the resistance of the (Na+ + K+)-ATPase to vanadium in intact cells.
Adenosine Triphosphate↗
Importance of extracellular divalent cations to polarisation of polymorphonuclear leukocytes induced by plasma.
The roles of extracellular calcium and magnesium ions in the polarisation of human peripheral blood polymorphonuclear leukocytes (PMN) induced by autologous fresh heparinised plasma were investigated by studying the effects of 5 mM chelators of divalent cations [ethylenediamine tetra-acetic acid (EDTA), ethylenebis-(oxyethylene-nitrilo)-tetra-acetic acid (EGTA) or disodium hydrogen citrate]. In addition, the effects of a blocker of membrane calcium channels (verapamil) were studied. Polarisation of PMN suspended in plasma (84.1 +/- 11.9%) was reduced by each chelating agent over 30 min (to 20.0 +/- 15.6% by EDTA, to 42.5 +/- 19.3% by EGTA and to 29.4 +/- 22.9% by citrate). Polarisation of PMN suspended in plasma treated with EDTA or EGTA was restored by inclusion of equimolar additional Ca2+ ions, and in plasma treated with EDTA, EGTA or citrate, by equimolar additional Mg2+ ions. Additional Mg2+ had no effect on the spherical shape of PMN in Hanks' solution and additional cations had no effects on the polarisation of PMN induced by fMLP. Cells rendered spherical by each chelating agent in plasma for 30 min retained their ability to polarise on addition of fMLP to the plasma-chelator medium. Verapamil (10(-4) M) markedly reduced polarisation in plasma (to 52 +/- 11.3%) but the same drug (10(-5) M) had no such effect. In contrast to the polarisation of cells in plasma, the polarisation response of PMN to N-formyl-methionyl-leucyl-phenylalanine (fMLP, 10(-8) M) in buffered Hanks' solution was not affected by any of the chelating agents or by verapamil, even in high concentration. These results indicate that extracellular divalent cations are necessary for the polarisation of PMN suspended in autologous plasma and that the mechanism of polarisation of PMN in plasma may be different to that of polarisation induced by fMLP.
Blood Physiological Phenomena↗
In vitro effect of mercury on tissue phosphatases of Notopterus notopterus and the role of EDTA and AA in their restoration.
In vitro effects of four sublethal concentrations (0.10, 0.40, 0.80 and 1.20 micrograms) of mercury (hg) on acid, alkaline and glucose-6-phosphatases of liver, kidney and gills of Notopterus notopterus were studied. The maximum (80.76%) significant (P less than 0.001) inhibition in acid phosphatase of gills and minimum (36.66%) significant (P less than 0.05%) inhibition in alkaline phosphatase of kidney was observed at 1.20 microgram and 0.10 microgram Hg concentrations, respectively. EDTA and ascorbic acid (AA) restored the phosphatase activity to the greatest extent when administered with the Hg.
Acid Phosphatase↗
Quantitation of reduced disulfide groups in monoclonal antibodies using 5-iodoacetamidofluorescein: a novel size exclusion-HPLC technique.
The interference of stannous in the quantitation of sulfhydryl in pretreated MAb using the Ellman's method is described. A HPLC method which effectively overcomes the interference by stannous ion in measuring sulfhydryls by the commonly used Ellman's technique has been developed. The method involves reacting the reduced monoclonal antibody with 0.1 M EDTA followed by the sulfhydryl specific reagent 5-iodoacetamidofluorescein and incubating the mixture at 37 degrees C for 1.0-1.5 h. The 5-IAF labeled protein is separated from the unbound free 5-IAF and its secondary degraded products by size exclusion HPLC using a TSK G3000 SWXL column. The method allows quantitation of MAb sulfhydryl present as monomeric MAb, aggregates and fragments and is a major improvement over the spectroscopic techniques.
Antibodies, Monoclonal↗
Enzymatic reduction of 5,5'-dithiobis-(2-nitrobenzoic acid) by lysate of rat liver mitochondria.
The oxidation of mitochondrial sulphydryl groups is known to increase the permeability of mitochondrial membranes and to be a key event in oxidative stress. Resistance to this damage is thought to involve thioredoxin reductase. In this study, the reduction of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) by a lysate of rat liver mitochondria was used to assay the mitochondrial disulphide reducing capacity. NADPH-dependent reduction of DTNB was used to distinguish enzymatic reduction from the non-enzymatic reduction. Enzymatic reduction by the mitochondrial lysate was suppressed by DTNB at concentrations exceeding 0.25 mM and by pH above 7.0. It was strongly inhibited by Zn2+ and Mn2+ (IC50 about 2.5 and 20 microM, respectively) and was more weakly inhibited by Mg2+ and Ca2+ (IC50 about 1.8 and 2.1 mM, respectively). As a consequence of inhibition by divalent cations, the reaction was stimulated by both physiological (ATP, ADP, pyrophosphate and citrate) and non-physiological (EDTA and EGTA) chelators. Reduction of insulin disulphides by the mitochondrial lysate was dependent on the presence of a divalent cation chelator during the isolation of mitochondria and during the enzyme assay. Our results suggest that stimulation of mitochondrial disulphide reducing activity by lowered pH, as well as by increased levels of ATP, ADP and citrate, has the potential to contribute to the maintenance of mitochondrial sulphydryl groups under oxidative conditions.
Adenosine Diphosphate↗
Pitfalls in the measurement of serum VEGF in children with congenital heart disease.
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Anticoagulants↗
Inhibition of laccase activity from Trametes versicolor by heavy metals and organic compounds.
Due to the numerous biotechnological applications of laccase enzyme, it is essential to know the influence of different agents usually present in the natural environment on its enzymatic action, especially for in situ treatment technologies. In the present work, a simple and rapid method to determine the inhibitory or inducer effect of different compounds on laccase activity was developed. The compounds tested were copper-chelating agents and heavy metals. It was found that using syringaldazine as a substrate, all copper-chelating agents (except EDTA) highly inhibited laccase activity (around 100%) at an inhibitor concentration lower than 20 mM. Moreover, 40% of inhibition, which was detected at a concentration of 20 mM for both Cd(2+) and Cu(2+) increased with concentration until nearly complete inhibition at 80 mM.
Basidiomycota↗
Enhanced phytoextraction of Pb and other metals from artificially contaminated soils through the combined application of EDTA and EDDS.
Chemically enhanced phytoextraction is achieved by the application of chelates to soils. Using pot experiments, the effect of the combined application of EDTA and EDDS on the uptake of Cu, Pb, Zn and Cd by Zea mays L. was studied. Among the tested application ratios of 1:1, 1:2, and 2:1 (EDTA/EDDS), 2:1 of EDTA:EDDS was the most efficient ratio for increasing the concentrations of Cu, Pb, Zn and Cd in the shoots. The combined application of 3.33 mmol kg(-1) soil of EDTA+1.67 mmol kg(-1) soil of EDDS produced 650 mg kg(-1) of Pb in the shoots, which was 2.4 and 5.9 times the concentration of Pb in the shoots treated with 5 mmol kg(-1) of EDTA and EDDS alone, respectively. The total phytoextraction of Pb reached 1710 microg kg(-1) soil, which was 2.1 and 6.1 times the total Pb from 5 mmol kg(-1) EDTA and EDDS alone, respectively. The combined application of EDTA and EDDS also significantly increased the translocation of Pb from the roots to the shoots. The mechanism of enhancing the phytoextraction of Pb by the combined application of EDTA+EDDS did not involve a change in the pH of the soil. The increase in the phytoextraction of Pb by the shoots of Z. mays L. was more pronounced than the increase of Pb in the soil solution with the combined application of EDTA and EDDS. It was thought that the major role of EDDS might be to increase the uptake and translocation of Pb from the roots to the shoots of plants.
Biodegradation, Environmental↗
The effect of operating variables on chelant-assisted remediation of contaminated dredged sediment.
The paper shows the results from a number of lab-scale washing treatments using the four chelating agents EDTA, NTA, citric acid and [S,S]-EDDS aiming at the remediation of a real heavy metal-contaminated sediment. Investigation of the influence of chelant type and concentration as well as solution pH was the major focus of the work. The analysis of speciation of metals and chelating agents in solution was carried out through geochemical speciation modelling in order to identify the optimal conditions for the washing process as well as to evaluate the competition phenomena of metal-chelant complexes in solution. The major competing cations were found to be Ca above all and Mg under specific conditions. Among the investigated chelating agents, EDDS appeared to be less affected by competition by major cations while ensuring adequate heavy metal extraction efficiencies. For a 1:1 chelant/metal ratio, the following ranking was observed: EDDS>Cit>NTA>EDTA for As, EDDS>NTA congruent withEDTA>>Cit for Cu, EDDS congruent withEDTA congruent withNTA>Cit for Zn, EDTA>NTA>EDDS>Cit for Pb at pH 5 and EDTA congruent withEDDS congruent withNTA>>Cit for Pb at pH 8. For a 10:1 chelant/metal ratio geochemical modelling indicated that at the equilibrium the extracting solutions were dominated by the free form of the chelating agents, indicating the inability of such species to complex trace metals due the strong interactions existing between heavy metal ions and sediment constituents.
Arsenic↗
Rapid separation of serum does not avoid artificially higher matrix metalloproteinase (MMP)-9 levels in serum versus plasma.
OBJECTIVES: To examine whether the time between blood drawing and centrifugation (TBDC) affects the levels of matrix metalloproteinase (MMP)-9 and MMP-2 levels in serum and in plasma samples, and to assess whether there is correlation between MMP-9 and MMP-2 levels in serum and plasma samples. DESIGN AND METHODS: Serum and plasma samples (N=8) were separated from venous blood collected into citrate, heparin, and EDTA tubes, which were either centrifuged immediately or after 5, 10, 20, or 30 min after blood drawing. We assessed the correlation between MMP-9/MMP-2 in serum and citrate, heparin, and plasma samples (N=20), which were assayed for gelatine zymography of MMP-2 and MMP-9. RESULTS: MMPs are released by platelets or leukocytes during platelet activation or sampling process, thus leading to artificially higher MMP-9 levels in serum compared with citrate, heparin, or EDTA plasma samples, independently of TBDC. Citrate and heparin plasma samples had the lowest Pro-MMP-9 and MMP-9 levels, which correlated with each other. Pro-MMP-9 levels in serum correlated with Pro-MMP-9 levels in EDTA or citrate plasma, but not with heparin plasma. While no significant correlations were found between MMP-9 levels in serum and those found in plasma samples, the total MMP-9 levels (Pro-MMP-9+MMP-9) in serum and in plasma samples correlated with each other. No significant differences were found in pro-MMP-2 levels. CONCLUSIONS: These results suggest that the circulating levels of MMP-9 should be assessed in citrate or heparin plasma samples, but not in serum samples because of artificially higher MMP-9 levels in serum, independently of TBDC, and because they do not correlate with the MMP-9 levels in plasma samples.
Citric Acid↗
Synthesis of pathogen inactivating nucleic acid intercalators.
A series of antiviral compounds consisting of an intercalating acridine derived part, a spacer region and a reactive EDTA-derived conjugate was synthesized in an easy sequence starting from 1,omega-alkyldiamines. As shown in model screenings, in the presence of ascorbic acid the Fe-complexes of these compounds reduced the phage-titer of MS2-phages by several logarithmic decades.
Acridines↗