Dithiobisnitrobenzoate Research Articles

Reactive Sulphydryl Groups present in Horse (Equus Ferus Caballus) Carbonmonoxyhaemoglobin by Titrating with 5, 5′‐Dithiobis‐2‐Nitrobenzoate (DTNB)

This research was carried out to determine the number of reactive sulphydryl groups in horse (Equus ferus caballus) haemoglobin. Haemolysate was prepared from the horse blood. The haemoglobin was separated into major and minor fractions using carboxymethyl cellulose (CMC‐52). Each haemoglobin fraction, in phosphate buffer pH 7.6, was accurately measured into several clean, dry test tubes. Increasing volumes of stock 5,5′‐dithiobis‐2‐nitrobenzoate (DTNB) were added to the different test tubes and left to equilibrate for 3 hours. Absorbance of each solution in each test tube was read at 412 nm. A graph of change in absorbance against the volume of DTNB was plotted; maximum change in absorbance was obtained at the point where the graph levels off. The ratios of 5‐thio‐2‐nitrobenzoate (TNB) concentration to the concentration of haemoglobin tetramer (Hb4) were calculated and plotted against the volume of DTNB. The number of sulphydryl groups reacting with DTNB was found to be two in both the major and minor haemoglobin.Support or Funding InformationCovenant University, Ota.

Identification of an essential cysteinyl residue for the structure of glutamine synthetase α from Phaseolus vulgaris

We have studied the possible role, in a plant glutamine synthetase (GS), of the different cysteinyl residues present in this enzyme. For this purpose we carried out the site-directed mutagenesis of the cDNA for alpha-GS polypeptide from Phaseolus vulgaris in the positions corresponding to Cys-92, Cys-159, and Cys-179, followed by heterologous expression in E. coli and enzymatic characterisation of WT and mutant proteins. The results show that neither Cys-92 nor Cys-179 residues were essential for enzyme activity, but the replacement of Cys-159 by alanine or serine strongly affects the quaternary structure and function of the GS enzyme molecule, resulting in a complete loss of enzymatic activity. Other studies using sulfhydryl specific reagents such as pHMB (p-hydroxymercuribenzoate) or DTNB (5,5'-dithiobis-2-nitrobenzoate) confirmed that the profound inhibition produced is associated with an important alteration of the quaternary structure of GS, and suggest that Cys-159 might be the residue responsible for the enzyme inhibition. All these results suggest that the Cys-159 residue is essential for the enzyme structure. The results are also consistent with previous reports based on classical biochemistry studies indicating the presence of essential cysteinyl residues for the enzyme activity of higher plant GS.

Modulation of [3H]Dopamine Release by Glutathione in Mouse Striatal Slices

Glutathione (gamma-glutamylcysteinylglycine, GSH and oxidized glutathione, GSSG), may function as a neuromodulator at the glutamate receptors and as a neurotransmitter at its own receptors. We studied now the effects of GSH, GSSG, glutathione derivatives and thiol redox agents on the spontaneous, K(+)- and glutamate-agonist-evoked releases of [(3)H]dopamine from mouse striatal slices. The release evoked by 25 mM K(+) was inhibited by GSH, S-ethyl-, -propyl-, -butyl- and pentylglutathione and glutathione sulfonate. 5,5'-Dithio-bis-2-nitrobenzoate (DTNB) and L-cystine were also inhibitory, while dithiothreitol (DTT) and L-cysteine enhanced the K(+)-evoked release. Ten min preperfusion with 50 microM ZnCl(2) enhanced the basal unstimulated release but prevented the activation of K(+)-evoked release by DTT. Kainate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) evoked dopamine release but the other glutamate receptor agonists N-methyl-D-aspartate (NMDA), glycine (1 mM) and trans-1-aminocyclopentane-1,3-dicarboxylate (t-ACPD, 0.5 mM), and the modulators GSH, GSSG, glutathione sulfonate, S-alkyl-derivatives of glutathione, DTNB, cystine, cysteine and DTT (all 1 mM) were without effect. The release evoked by 1 mM glutamate was enhanced by 1 mM GSH, while GSSG, glutathionesulfonate and S-alkyl derivatives of glutathione were generally without effect or inhibitory. NMDA (1 mM) evoked release only in the presence of 1 mM GSH but not with GSSG, other peptides or thiol modulators. L-Cysteine (1 mM) enhanced the glutamate-evoked release similarly to GSH. The activation by 1 mM kainate was inhibited by S-ethyl-, -propyl-, and -butylglutathione and the activation by 0.5 mM AMPA was inhibited by S-ethylglutathione but enhanced by GSSG. Glutathione alone does not directly evoke dopamine release but may inhibit the depolarization-evoked release by preventing the toxic effects of high glutamate, and by modulating the cysteine-cystine redox state in Ca(2+ )channels. GSH also seems to enhance the glutamate-agonist-evoked release via both non-NMDA and NMDA receptors. In this action, the gamma-glutamyl and cysteinyl moieties of glutathione are involved.

Unique properties of NADP-thioredoxin reductase C in legumes

NADP-thioredoxin reductases (NTRs) reduce thioredoxins (Trxs), using NADPH as a reductant, together constituting complete redox systems (NTS). Beside NTRA and NTRB targeted to both cytosol and mitochondria of plant cells, there is in chloroplasts an unusual NTR (NTRC) harbouring a Trx domain in a C-terminal extension, as recently reported in Oryza sativa. Although NTRC may constitute a complete NTS, it was described as a bifunctional enzyme. Because the gene is only present in photosynthetic organisms and the protein in green tissues, NTRC was thought to have a role restricted to photosynthetic cells. To determine whether NTRC from dicot plants is a bifunctional enzyme or a complete NTS, as well as to identify its putative target, NTRC from Medicago truncatula was cloned and NTRA was cloned for comparison. Here evidence is presented that MtNTRC (i) acts as an NTS and reduces dithiobisnitrobenzoate (DTNB) with a turnover (0.62 s(-1)) similar to that measured with MtNTRA in the presence of a Trxh (0.81 s(-1)); (ii) is able to use both NADPH (k(M)=2.4 microM) and NADH (k(M)=11 microM) as cofactors; (iii) efficiently reduces BAS1, a plastidial peroxiredoxin; and (iv) is expressed in both leaves and stems but unexpectedly is even more abundant in cotyledons from dry and germinating seeds. Because BAS1 is also present in both green tissues and seeds, NTRC/BAS1 may be involved in the scavenging of peroxides produced in green tissues during the day or the night and in seeds during germination. These results suggest different roles for NTRC in monocot and dicot plants.

Molar absorption coefficients for the reduced Ellman reagent: reassessment

The Ellman method for assaying thiols is based on the reaction of thiols with the chromogenic DTNB (5,5 ′-dithiobis-2-nitrobenzoate) whereby formation of the yellow dianion of 5-thio-2-nitrobenzoic acid (TNB) is measured. The TNB molar absorption coefficient, 13.6×10 3 M −1 cm −1 , as published by Ellman in 1959 has been almost universally used until now. Over the years, however, slightly different values have been published, and it has further been shown that TNB reveals thermochromic properties. This should be taken into account when the Ellman method is used for determination of enzyme activities, such as in cholinesterase assays. Our data show that the absorbance spectra of TNB are shifted to longer wavelengths when temperature increases, while absorbance maxima decrease. Our recommended molar absorption coefficients at 412 nm are 14.15×10 3 M −1 cm −1 at 25 °C and 13.8×10 3 M −1 cm −1 at 37 °C (0.1 M phosphate buffer, pH 7.4). Molar absorption coefficients for other temperatures and wavelengths are included in the paper.

A Sulfenic Acid Enzyme Intermediate Is Involved in the Catalytic Mechanism of Peptide Methionine Sulfoxide Reductase fromEscherichia coli

Methionine oxidation into methionine sulfoxide is known to be involved in many pathologies and to exert regulatory effects on proteins. This oxidation can be reversed by a ubiquitous monomeric enzyme, the peptide methionine sulfoxide reductase (MsrA), whose activity in vivo requires the thioredoxin-regenerating system. The proposed chemical mechanism of Escherichia coli MsrA involves three Cys residues (positions 51, 198, and 206). A fourth Cys (position 86) is not important for catalysis. In the absence of a reducing system, 2 mol of methionine are formed per mole of enzyme for wild type and Cys-86 --> Ser mutant MsrA, whereas only 1 mol is formed for mutants in which either Cys-198 or Cys-206 is mutated. Reduction of methionine sulfoxide is shown to proceed through the formation of a sulfenic acid intermediate. This intermediate has been characterized by chemical probes and mass spectrometry analyses. Together, the results support a three-step chemical mechanism in vivo: 1) Cys-51 attacks the sulfur atom of the sulfoxide substrate leading, via a rearrangement, to the formation of a sulfenic acid intermediate on Cys-51 and release of 1 mol of methionine/mol of enzyme; 2) the sulfenic acid is then reduced via a double displacement mechanism involving formation of a disulfide bond between Cys-51 and Cys-198, followed by formation of a disulfide bond between Cys-198 and Cys-206, which liberates Cys-51, and 3) the disulfide bond between Cys-198 and Cys-206 is reduced by thioredoxin-dependent recycling system process.

Role of Cysteine in Activation and Allosteric Regulation of Maize Phosphoenolpyruvate Carboxylase

The effect of 5-5'-dithiobis-2-nitrobenzoate (DTNB) on the kinetic parameters and structure of phosphoenolpyruvate carboxylase purified from maize (Zea mays L.) has been studied. The V(max) is found to be independent of the presence of this thiol reagent. The K(m) is increased upon oxidation of cysteines by DTNB. At a substrate concentration higher than K(m) (3.1 millimolar Mgphosphoenolpyruvate), a significant reversible decrease of the activity is observed. Malate has little effect in preventing the modification of these cysteines. The V type inhibition by malate was also studied at a saturating phosphoenolpyruvate level (9.3 millimolar Mgphosphoenolpyruvate). In the presence of 50 micromolar DTNB, up to 60% inhibition is caused by 15 millimolar malate; however, in the presence of both 50 micromolar DTNB and 50 millimolar dithiothreitol (DTT) this inhibition is reduced to 20%. The presence of DTT alone increases the size of the phosphoenolpyruvate carboxylase molecule as determined by light scattering. The activity at nonsaturating substrate concentration is increased by 36% in the presence of DTT. The oligomerization equilibrium between the dimer and the tetrameric form of the enzyme is affected by cysteine. The K(m) for the substrate, the sensitivity toward malate, and the size of the enzyme are found to be modified upon incubation in the presence of DTT.

Mapping of fish myosin light chains by two-dimensional gel electrophoresis

1. 1. Myosins were prepared from the ordinary muscle of 16 fish species as well as from rabbit fast muscle, and light chain subunits [alkali light chains A1, A2 and DTNB (5,5′-dithio-bis-2-nitrobenzoate) light chain] were separated on two-dimensional gel electrophoresis in combination with isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 2. 2. A1 light chains showed mol. wts ranging from 21,000 to 22,900 and isoelectric points ranging from 4.51 to 4.62. DTNB light chains were spotted in a narrow area, with a mol. wt range of 16,800–17,600 and an isoelectric point range of 4.48–4.55. On the other hand, A2 light chains were most species-speciic, with a mol. wt range of 14,000–19,500 and an isoelectric point range of 4.31–4.46. 3. 3. It was suggested that the lower species-specificity in A1 as opposed to A2 is accounted for by the addition of an N-terminal peptide (“difference peptide”) in the former. The properties and possible role of this peptide are discussed.

Reactivities of sulfhydryl groups in native and metal-free aminoacylase I.

Aminoacylase I from porcine kidney (EC 3.5.1.14) contains seven cysteine residues per subunit. Three sulfhydryl groups are accessible to modification by 4-hydroxymercuribenzoate (p-MB). The kinetics of the reaction suggest that only one of these groups affects acylase activity when modified by p-MB. Its reaction rate increases 2-3-fold when the essential metal ion of aminoacylase is removed. Modification of metal-free apoenzyme by N-ethylmaleimide (NEM) abolishes its activity without impairing Zn2+ binding. This indicates that the sulfhydryl group reacting with NEM is not directly coordinated to the metal. DTNB (5,5'-Dithio-bis(2-nitrobenzoate), Ellman's reagent) also modifies three sulfhydryl groups per subunit. In this case, the reactivities of native aminoacylase and apoenzyme are not significantly different. N-Hydroxy-2-aminobutyrate, a strong aminoacylase inhibitor, substantially increases the reactivity of the slowest reacting sulfhydryl in both native enzyme and metal-free aminoacylase. It appears that binding of the inhibitor or removal of the metal ion induces conformational changes of the amino-acylase active site that render a buried sulfhydryl group more accessible to modification.

Chemical modification of thiol group(s) in protein: Application to the study of anti-microtubular drugs binding

1. 1. Different chemical procedures such as performic oxidation, carboxymethylation, carboxyethylation, aminoethylation, cyanylation, acylation, arylation etc. and addition of thiols to activated double bonds, titration of thiols with DTNB (Dithiobis-Nitro-Benzoate) and the reaction of thiols with organomercurials and the titration with p-chloro-mercuri-benzoate (PCMB) etc. are cited and discussed. Their chemical reactions are shown in the figures. 2. 2. We describe in this paper that several chemicals interfere with microtubule assembly by combining with sulfhydryl residues. Reagents such as Cytochalasin-A and B, ethylacetylacrylate, FDNB (fluorodinitrobenzene), NEM ( N-ethyl-maleimide), diamide, EBI (ethylene-bis-iodoacetamide, ethacrynic acid, methal ions, methylmercury, triethyllead ion and CDDP (cis-dichlorodiammine-platinium-II) are cited and their mechanisms are discussed.

Purification and characterization of bolaffinine, a toxic protein from Boletus affinis Peck (Boletaceae)

A toxic protein of Mr 22,000, called bolaffinine, has been purified from the mushroom, Boletus affinis Peck (Boletaceae), with a 0.65% yield using a procedure involving four steps: ammonium sulfate precipitation, two chromatographies on ion-exchange columns and gel filtration on Sephadex G-75. It is thermolabile and can be inactivated by organic solvents. Its isoelectric point lies between pH 9 and 10. It contains 234 amino acids, no free SH groups and one disulfide bridge as evidenced by reactions with 5-5'-dithiobis-nitrobenzoate (DTNB) and 2-mercaptoethanol. When injected into mice, it provokes the death of animals within 16-24 h. The 24 h LD50 is 61 mg/kg of body weight. Bolaffinine inhibits protein synthesis in hepatoma tissue culture and in a rabbit reticulocyte lysate.

The characterization of an adenosine deaminase from chicken serum

1. 1. Adenosine deaminase from chicken serum was purified and characterized. 2. 2. The K m and V max values of the enzyme in respect of adenosine as substrate were 7.14 × 10 −3 M and 8.5 μM/min respectively. AMP, ADP & ATP are poorly metabolized respectively in decreasing order. 3. 3. The enzyme had no NA Dase and NADPase activities but the presence of either NAD or NADP in normal incubation mixture activated the enzyme. 4. 4. Metal ions Zn 2+, Ca 2+ and Mg 2+ activated the enzyme to various levels. 5. 5. Some compounds, mercaptoethanol, dithiothreitol (DTT), dithiobisnitrobenzoate (DTNB) inhibited the enzyme to various extents.

The reactivity of sulfhydryl groups of yeast DNA dependent RNA polymerase I.

The number of reactive cysteine residues of yeast RNA polymerase I was determined and their function was studied using parachloromercury benzoate (pCMB), dithiobisnitrobenzoate (DTNB) and N-ethyl-maleimide (NEM) as modifying agents. By treatment with DTNB about 45 sulfhydryl groups react in the presence of 8M urea. Under non-denaturing conditions only 20 sulfhydryl groups are reactive with pCMB and DTNB. Both reagents completely inactivate the enzyme and this effect can be reversed by reducing agents. The sedimentation coefficient and the subunit composition are not affected when the enzyme is inactivated. Two of the most reactive sulfhydryl groups are necessary for activity. The modification of these groups is partially protected by substrates and DNA, suggesting that they are involved either in catalysis or in the maintenance of the conformation of the active site. Experiments with 14C-NEM indicate that the most reactive groups are located in subunits of 185,000, 137,000 and 41,000 daltons.

Purification and some properties of formate dehydrogenase.

Purified formate dehydrogenase was prepared from dry seeds of pea (Pisum sativum). The enzyme preparation was more than 95% homogeneous in terms of acrylamide-gel disc electrophoresis and the molecular weight was found to be 70,000±2,000 by gel filtration and 72,000±2,000 by sedimentation equilibrium. The pH-activity curve, spectral properties, and amino acid composition were investigated. The enzyme was colorless and contained no significant amounts of iron, manganese, and zinc. Four moles of PCMB (p-chloromercuribenzoate) reacted with the enzyme and the activity of the enzyme was almost completely inhibited as a result. The reaction with PCMB was retarded in the presence of NAD+. On the other hand, DTNB (dithio-bis-nitrobenzoate) was found to react with sulfhydryl groups of the enzyme much faster in the presence of NAD+. Approximately 3.5 moles of sulfhydryl groups per enzyme reacted with DTNB in the presence of NAD+ but the modified enzyme still retained 70% of its original activity. The addition of sodium dodecylsulfate caused further reaction of the enzyme with DTNB and 8 moles of sulfhydryl groups per enzyme were detected in total by the DTNB method.

69] Purification of an enzyme containing a NADP-protected cysteine on organomercuri-agarose 17β-estradiol dehydrogenase

Publisher Summary 17 β-Estradiol dehydrogenase, among other enzymes, possesses essential cysteinyl residues which are protected by the coenzyme against thiol reagents, such as p-hydroxymercuribenzoate (PMB), N-ethylmaleimide (NEM), or 5, 5'-dithiobisnitrobenzoate (DTNB). The incubation of an impure enzyme solution with thiol reagents in the presence of NADP results in the reaction of all cysteinyl residues that are not protected by this coenzyme. After removal of both NADP and exogenous thiol reagents, only the unmodified residues are able to react with an organomercuri-Sepharose derivative. These proteins are readily eluted by β-mercaptoethanol or dithiothreitol. The method provides the basis of a successful purification procedure if activity is retained.