2,2 -Dithiobis method

Mann and Mitchell [58] described a simple colorimetric method for estimation of (-D)-penicillamine in plasma. Blood containing 2-50 pg of penicillamine was mixed with 0.1 M EDTA solution in tromethamine buffer solution. 0.1 mL of this solution was adjusted to pH 7.4 and centrifuged. To a portion of the plasma was added 3 M HCL, the mixture was freeze-dried, and a suspension of the residue in ethanol was centrifuged. The supernatant liquid was mixed with tromethamine buffer solution (pH 8.2) and 10 mM 5,5 -dithiobis-(2-nitrobenzoic acid) in phosphate buffer solution (pH 7.0), the mixture was shaken with ethyl ether, and the absorbance of the separated aqueous layer was measured at 412 nm. The mean recovery was 60% (four determinations), and the calibration graph was linear for the cited range. 

Method A. The rationale of method A is that HDL and LDL are separated by selective precipitation of LDL by dextran sulfate and Mg2+ after the reaction between LDL and reconstituted HDL containing radiolabeled CE by CETP. The method was originally described by Kato et al. [77], The assay mixtures consist of reconstituted [14C]CE-HDL as the donor for CE, LDL as the acceptor, 5,5 -dithiobis-2-nitrobenzoic acid, bovine serum albumin (BSA), partially purified CETP, and a test sample in Eppendorf tubes (1.5 ml). After a 30-min incubation at 37°C, the reaction is terminated by the addition of an LDL-precipitation solution. After standing for 20 min in an ice bath, the assay mixtures are centrifuged, and the supernatant solution containing [14C]CE-HDL is analyzed for radioactivity. Furthermore, the [14C]CE-LDL precipitate is also analyzed for radioactivity if necessary. Usually the blank and control transfer values are about 6% and 34% of initial [14C]CE-HDL added under the assay conditions, respectively. 

The method is based on the partitions of the anions to a cationic micelle phase and shows different selectivity from ion exchange chromatography In a kinetic method [13] for the determination of thiocyanate, sulphite and sulphide the anions are reacted with 5,5 dithiobis (2-nitrobenzoic acid) in aqueous cetyltrimethyl/ammonium bromide micelles. 

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). 

Citrate Synthetase. The assay method is modified from that of Srere, Brazil, and Gonen. The reaction mixture contains, in a volume of 1.3 ml, 7.7 X 10 M Tris (adjusted to pH 8.0), 1.5mM 5,5 -dithiobis-(2-nitrobenzoic acid), l.lmM MgCU, 2.6mM oxalacetate, 1.8 x lO M acetyl-CoA, and from 2 to 25 ig of protein, depending on which fraction of gradient is being assayed. The assay is initiated by the addition of the... 

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). 

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 method of Fan and Dasgupta (1994) relics on tlie reaction of formaldehyde with 1,3-cyclohexane-dione in acidified ammonium acetate to form the fluorescent dihydropyridine derivative in a flow injection analysis system. Formaldehyde trapped in water can be reacted with pararosaniline and sodium sulfite under mild conditions (neutral pH, room temperature equilibration) to produce a colored product that is measured at 570 nm (Petreas et al. 1986). The presence of bisulfite is an interference in this reaction so the method cannot be used to sample atmospheres that contain sulfur dioxide. In addition, the method is reported to suffer from interferences resulting from the presence of other aldehydes and phenol (Hoogenboom et al. 1987). The indirect method of Hoogenboom et al. (1987) relies on the reaction of excess bisulfite in an aqueous solution of formaldehyde with 5,5 -dithiobis(2-nitrobenzoic acid) to form a colored product, the absorbance of which is measured at 412 nm. The method reported by Naruse et al. (1995) relies on the formation of a colored product obtained by reacting the aqueous formaldehyde with acetylacetone and ammonium acetate in acetic acid. Absorbance is measured at 414 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... 

D16. Dietz, A. A., Rubinstein, H. M., and Lubrano, T., Colorimetric determination of serum cholinesterase and its genetic variants by the propionylthiocholine-dithiobis(ni-trobenzoic acid) procedure. Sel. Methods Clin. Chem. 8, 41-46 (1978). 

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. 

Amino-l,2-benzisothiazoles were prepared in satisfactory yield by reaction of 2,2 -dithiobis(benzonitrile) with various secondary amines followed by oxidation <978871>. This method was found to be superior to other methods of preparation, which had usually involved nucleophilic displacement of a 3-halobenzisothiazole as the last step. This reaction t)fpically gave moderate yields of aminobenzisothiazole due to the isothiazole s ring lability with nucleophiles. 

A very sensitive and commonly used method for cholinesterase determination was described by Ellman et al. (E2), based on hydrolysis of the thiocho-line 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 forming... 

Serum pseudo-cholinesterase (referred to hereafter as serum cholinesterase) activities were determined from a five minute reaction period at room tenperature with a modification of the method of EUman et 2Q. ( ). Briefly, a 10 pi 2d.iguot of serum ves added to both a reference and sample cuvette ocxitaining 3.0 ml of 5,5-dithiobis-(2-nltrobenzoate) (DDO buffer (0.25 mM in ph 8.0, 0.1 M sodium phos te buffer) and mixed with a micro-stirring rod. Die reaction ves steurted in the sample cuvette by the addition of 20 pi of aoetylthiochollne iodide (78 nM) and stirred. The reference cuvette received 20 pi of distilled water in place of substrate. Absorbance was measured at 412 im over a five minute period and the rate calculated from the slope of the derived curve. 

Alkaline phosphatase has been reversibly immobilized on cross-linked agarose by two methods, both based on disulphide linkages.Prior to coupling with 5,5 -dithiobis-(2-nitro-benzoic acid)-activated thiol-agarose (57), the enzyme was modified by thiolation with 4-mercaptobutyrimidate. The unmodified enzyme could be coupled directly to dithiobis(succinimidyl propionate)-agarose (58). 

Preparative Methods 2-allyloxybenzothiazoles are prepared by reaction of potassium allyloxides with 2-chlorobenzothiazole in dry ether at rt. 2-Allylthiobenzothiazoles are prepared by reaction of allylic alcohols with 2,2 -dithiobis(benzothiazole) and triphenylphosphine or with benzothiazole-2-thiol, diethyl azodicarboxylate, and triphenylphosphine in dry toluene at rt. ... 

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