Dithiobis additive

Additional information <1-4, 11> (<3> not inactivated by 5,5 -dithiobis(2-nitrobenzoic acid), tetranitromethane or 2-hydroxy-3-nitro-benzyl bromide... 

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. 

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... 

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. 

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. 

Recent work with aurodox (86) has included study of the interaction of 86 with EF-Tu and its total synthesis. The interaction of 86 with EF-Tu has been studied using fluorescence emission spectroscopy. The reaction of EF-Tu in the presence of 86 with l-anilino-8-napthalenesulfonate [or 5,5 -dithiobis(2-nitrobenzoate)] was found similar to that of EF-Tu-GTP, supporting the formation of a GTP like conformation for the EF-Tu-GDP-86 complex [248]. In addition, conformational changes to alter the environment around the sole tryptophan residue of EF-Tu were observed [248], Incorporation of 3-fluorotyrosine into EF-Tu and subsequent l F nmr studies indicated that the conformation of EF-Tu-86 was similar to that of EF-Tu-GDP-EF-Ts and EF-Tu-GTP, and quite different from EF-Tu-GDP [249], In another study, 86 was found to increase the affinity of EF-Tu-GDP for aa-tRNA, and to decrease the affinity of EF-Tu-GTP [250]. The first total synthesis of 86 and 87 has been reported [252,253]. 

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. 

The additional redox chromophore in IfFd was traced to the Cys(V), Cys(VI) pair by titrations with 5,5 -dithiobis(2-nitrobenzonic acid) (DTNB), which revealed a complete redox cycle of P/4Fd is shown schematically in Fig. 12. Hence the subscript A and B to the Fd with oxidized or reduced cluster refer to the oxidation state of the Cys(V), Cys(VI) pairs, with subscript A denoting an intact disulfide and subscript B denoting free thiols. The H NMR spectra of the 3Fe Fd and 3Fe Fd have been similarly characterized. ... 

To stabilize potentially weak or transient protein interactions, whole, transfected cells can be subjected to chemical cross-linking prior to lysis. For cross-linking, the cells were incubated for 1 h at room temperature with the membrane-permeant, cleavable, homobifunctional cross-linker dithiobis (succinimidylpropionate) (DSP) (Pierce Chemical Co., Rockford, IL) (1 mM final concentration in PBS). Cross-linking was stopped by the addition of Tris-HCl, pH 7.7 to a 10 mM final concentration. After immunoprecipitation, the beads are boiled in SDS-sample buffer containing 5 % ) -mercaptoethanol to cleave the cross-linker. This approach has been used to study DLPl interactions on the mitochondrial membrane (Yoon et al., 2003). 

Analysis of Reagent Purity the presence of monothiols can be detected by addition of arsenite (which forms a tight tridentate complex with DTT), followed by addition of 5,5 -dithiobis-(2-nilrobenzoate). Total thiol content is determined by omitting arsenite. 

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