Dithiobis 2-nitrobenzoic acid DTNB

The DTNB Reaction of Metallothionein. The appearance of reactive (or free ) thiols in the 10,000 Dalton peak of rat renal cytosol, which was previously reported, was apparently inconsistent with the published properties of equine renal metallothionein—namely, that all sulfhydryl groups were present as metal mercaptide linkages. We have subsequently examined the reaction of 5,5 -dithiobis(2-nitrobenzoic) acid (DTNB, a standard agent for measurement of the SH concentrations ( )) with Zn-thionein. A bi-phasic reaction was found in which each step had DTNB dependent kr = 0.41 M , sec  

The biphasic nature of the reaction will probably require elucidation of the tertiary structure of MT for a correct explanation. 

For our purposes, the significant aspect of this reaction is that it provides an explanation for the reactive SH groups of the 10,000 Dalton fractions of the kidney cytosol they are the result of metallothionein reacting slowly with the DTNB. The measured rate constants are adequate to explain the 50 yM SH concentration previously observed after fifteen minutes reaction time. 

ACS Symposium Series American Chemical Society Washington, DC, 1980. 

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Savas, M.M., Petering, D.H. and Shaw, C.F. Ill (1991) On the rapid, monophasic reaction of the rabbit liver metaDothionein a-domain with 5,5 -dithiobis (2-nitrobenzoic acid) (DTNB). Inorganic Chemistry, 30, 581-583. 

Ellman s reagent 5,5 -dithiobis(2-nitrobenzoic acid DTNB) reacts quantitatively with - SH groups (Eq. 3-41) to form mixed disulfides with release of a thiolate anion that absorbs light at 412 nm with a molar extinction... 

Probably the most frequently used spectrophotometric method to detect thiol groups, both for non-protein and protein sulphydryl groups involves the use of Ellman s reagent (Scheme 7.5). 5,5 -Dithiobis(2-nitrobenzoic acid), (DTNB) (Ellman 1959) undergoes disulphide exchange with thiol groups and the formation of 5-thio-2-nitrobenzoate anion (TNB) (Scheme 7.6). 

In brief, the method involves the glutathione reductase-catalyzed reduction of GSSG by NADPH, followed by the reaction of GSH with Ellman s reagent, (5,5 -dithiobis-2-nitrobenzoic acid DTNB). The chromophoric product, TNB, has an absorbance between 405 -12 nm. The reaction can therefore be followed spectrophotometrically (Reaction 3). 

Virtually all of the nonprotein sulfhydryl groups of RBCs are in the form of reduced GSH. 5,5 -Dithiobis(2 nitrobenzoic acid) (DTNB) is a disulfide chromogen that is readdy reduced by sulfhydryl compounds to an intensely yellow compound. The absorbance of the reduced chromogen is measured at 412 nm and is directly proportional to the GSH concentration. 

An interesting derivatization method for cysteine was recently described by Jenke and Brown [35], They mixed the column effluent with a buffered solution of 5,5 -dithiobis(2-nitrobenzoic acid) (DTNB), yielding a strongly yellow-colored chromophore, which can be detected photometrically at 412 nm ... 

The colorimetric method is based on the hydrolysis of the substrate acetylthiocholine to acetate and thiocholine as performed by the cholinesterase. Thiocholine is then reacted with 5,5 -dithiobis(2-nitrobenzoic acid) (DTNB) to form a yellow anion (5-thio-2-nitrobenzoate). The latter is quantitated by spectrometric analysis at 405 nm, with the concentration being proportional to the cholinesterase activity in the given sample. Also for a few days postmortem the cholinesterase activity in different tissues is measurable. ... 

A procedure for identifying certain cholinesterase variants was proposed by Dietz et al. (D15). After a period during which comments and discussion were offered by others working in the field, the method was published in Selected Methods of Clinical Chemistry (D16). This method is based upon the Ellman reaction (ElO), which was used by Ellman et al. (Ell) for the assay of acetylcholinesterase, and by Garry and Routh (G9) for the assay of serum cholinesterase. In these assay procedures, a thiocholine ester is used as the substrate. The thiocholine produced upon hydrolysis reacts with 5,5 -dithiobis(2-nitrobenzoic acid) (DTNB) to yield 5-thio-2-nitrobenzoate anion and other products. The rate of the reaction may be determined by measuring the rate at which... 

Thioredoxin reductase from calf liver was found to reduce 5,5 -dithiobis-(2-nitrobenzoic acid) (DTNB) at the expense of NADPH even in the absence of thioredoxin as an intermediate electron carrier, indicating that this mammalian reductase may have a wider substrate specificity than the corresponding enzymes from E. coli and yeast. 

A simple method to measure the membrane permeability to specific molecules has been presented by G. Battaglia and coworkers [141], The authors encapsulated highly hydrophilic 3,3, 3//-phosphinidynetris-benzenesulfonic acid (PH) into polyethylene oxidc)-co-poly(butylene oxide) (EB) vesicles and monitored its reaction with 5,5/-dithiobis-2-nitrobenzoic acid (DTNB) penetrating the membrane from the exterior. The reaction rate (amount of the formed product as a function of time after DTNB addition) measured with IJV/Vis was directly correlated to the permeability of the permeating molecule. A comparison of these results with the permeability of egg yolk phosphatidylcholine (PC) vesicles showed that EB membranes have a more selective permeability toward polar molecules than the phospholipids membranes. Also in this case the permeability appeared to depend on the membrane thickness as predicted by Fick s first law. 

Acetylcholinesterase (AChE) The lyophilized enzyme from electric eel (Sigma) was dissolved in 0.05M phosphate buffer (pH-7.4) at a concentration of 20 pg/mL. Acetylthiocholine iodide was used as substrate at a final concentration of 5x10 4M in buffer. 5,5 -Dithiobis-(2-nitrobenzoic acid) (DTNB) at a final concentration of 3.8xlO 2M was used to monitor the released thiocholine according to a published procedure (41) with slight modification. Acetone was used as a solvent for the inhibitors. 

A radiometric and a spectrometric assay have been developed to measure PhaC activity. The radiometric assay measures the incorporation of isotope-labeled hydroxyacyl moieties into the polyester, which is present from the beginning as primer [17]. [3-14]ft-(-)-3-hydroxybutyryl-CoA or [3H]-P,S-3-hydroxybutyryl-CoA or in principle any other CoA thioester of a radioactively-labeled hydroxyacyl moiety could be used as substrate. Only the radioactivity that is really incorporated into the insoluble polyester is measured. The time course of the assay, the need to synthesize the substrates and the high costs make the assay very inconvenient and it is hardly used anymore. A more convenient assay is the spectrometric assay which measures the release of coenzyme A during the polymerization reaction in presence of Ellmann s reagent 5,5 -dithiobis-(2-nitrobenzoic acid) (DTNB) yielding 5 -thio(2-nitrobenzoate) that absorbs at about 412 nm [16], Here, the enzyme activity is measured directly without delay. However, it is not the formation of the polymeric product that is measured, but the release of coenzyme A, which can also be due to the hydrolytic cleavage of the substrate by another enzyme that does not have any PhaC activity, like a thioesterase. Nevertheless, this assay is now most frequently used due to its convenience. 

Ellman s reagent, 5,5 -dithiobis(2-nitrobenzoic acid) (DTNB), produces 2-nitro-5-sulfhydrylbenzoic acid by the reaction with thiols, which shows visible absorption at 412nm. Acetyl-CoA thioester is separated by ion-pair LC followed by conversion to thio-CoA with a postcolumn immobilized enzyme column reactor of phosphotransacetylase. Thio-CoA thus liberated can be determined spectrophotometri-cally after reaction with Ellman s reagent with the detection limit of 0.05 nmol. 

Quantitative analysis of the effect of (CTA)B micelles and (DODA)C vesicles on the rate of decomposition of 5,5 -dithiobis(2-nitrobenzoic acid) (DTNB), using pseudophase models, has been described [19]. The maximum acceleration by vesicles is 1500 fold and was ascribed, essentially, to reagent concentration at the vesicle bilayer [19]. 

Purified cottonseed NAPE synthase enzyme exhibited non-Michaelis-Menten biphasic kinetics with respect to the free fatty acid substrates, palmitic and linoleic acids. Kinetic parameters for the two saturable sites were calculated from various transformations e.g., double-reciprocal and Hill plots Cornish-Bowden, 1995) of initial velocity/ substrate concentration data and are summarized in TABLE 1. Preliminary experiments with several group-specific modifiers indicated that NAPE synthase was progressively inactivated by increasing concentrations of 5,5 -dithiobis(2-nitrobenzoic acid) (DTNB), diisopropyl fluorophosphate (DFP), phenylmethylsulfonylfluoride (PMSF), diethylpyrocarbonate (DEPC) (TABLE 2). These results suggest that NAPE synthase may form a thioester- or ester-intermediate through a cysteine or serine residue, respectively, and a histidine residue may participate in catalysis as well. 

The polymer-bound lipoic acid can be reduced by using NaBH4 (see Scheme 15-5). The reduced polymers are stable between pH 2-10 and can be reused after reduction. The resulting reduced polymer bisthiol is a potential reducing agent for disulfides. The degree of lipoyl substitution in the polymer can be found by reduction of 5,5 -dithiobis-(2-nitrobenzoic acid) (DTNB) (Ellman, 1959). The thiol content of the polymer can also be determined by reaction of the polymer with iodo[ C]acetic acid and by measurement of the uptake of radioactivity by liquid scintillation counting. 

Ellman s assay was first described " in 1959 and has undergone many minor modifications over the years. " The procedure is based on the reaction of thiols with Ellman s reagent or 5,5 -dithiobis-(2-nitrobenzoic acid) (DTNB) to yield a modified protein thiol (mixed disulfide), and a molecule of 2-nitro-5-thiobenzoic acid (TNB) as shown in below (Figure 2). 

A very sensitive and commonly used method for ChE determination was described by Ellman et al. (1961). This method uses thiocholine esters as substrates (acetyl- and butyrylthiocholine or others). After enzymatic hydrolysis, the relevant acid and thiocholine are released and thiocholine by its SH-group is detected using 5,5 dithiobis-2 nitrobenzoic acid (DTNB) forming 5-mercapto-2-nitrobenzoate (3-thio-6-nitrobenzoate, TNB) with an absorbance maximum of approximately 412nm (Figure 65.1). 

Nekrassova et al. [13] have also studied the electrochemical oxidation of 5-thio-2-nitrobenzoic acid (TNBA) at a BDD electrode for the indirect detection of cysteine, homocysteine and glutathione. The reaction was shown to proceed via a CEC reaction process at lower pH, in which the thiol moiety of the TNBA species must undergo deprotonation before oxidation. These authors also reported that 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) can be used to develop the total thiol detection methodology using chronoamperometry with BDD electrodes. The detection limits of cysteine, homocysteine and glutathione were found to be 5.7 pM, 4.4 pM and 5.8 pM respectively. 

LCAT, by consuming cholesterol, promotes its net transfer from non-hepatic cells into plasma and from other lipoproteins to the site of esterification. Evidence comes from experiments in vitro as well as in vivo. LCAT can be inhibited in vitro by compounds such as dithiobis (2-nitrobenzoic) acid (DTNB). Under these conditions net transport of cholesterol from cells ceases. In patients with a genetic deficiency of LCAT, free cholesterol accumulates in cells. Plasma from such people is ineffective in promoting net transport of cholesterol from cellular membranes. One product, cholesteryl ester, is redistributed among plasma lipoproteins. The other, lysophosphatidylcholine, is transferred to albumin from which it is rapidly removed and recycled. 

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