Mercaptoethanol and Dithiothreitol

Despite the assertion over a number of years that vanadate is rapidly reduced in the presence of ligands such as p-mercaptoethanol and dithiothreitol, it is now known that this assertion is subject to qualification. Solution studies have shown that the vanadium(V) complexes of p-mercaptoethanol [36] and dithiothreitol [37] can be quite stable in solution. At pH 7.1, reduction of vanadium in the dithiothreitol complex occurs in a timescale of about 90 min, and even at pH 6.2, significant production of V(IV) is not observed for about 20 min. When the crystalline vana-dium(V) complex of p-mercaptoethanol was dissolved in water, it was shown to 

Dithiothreitol is a more complex ligand than p-mercaptoethanol, and there is a corresponding increase in complexity of the solution chemistry. Two binuclear products (-352, -362 ppm -399, -526 ppm), both of V2L stoichiometry, have been identified. The individual vanadiums of each product are chemically distinct. The NMR studies [37] show that in one of the complexes, both vanadiums have a single coordinated sulfur. In the second complex, one of the two vanadiums has sulfur coordinated, whereas the second of the two has only oxygen in the coordination sphere. Neither of these compounds have been characterized by x-ray diffraction 

3 Cysteine, Glutathione, Oxidized Glutathione, and Other Disulfides 

Cysteine is a fairly effective reducing agent for vanadate, but even so, under neutral conditions, the V(V) lifetime is sufficiently long that vanadium NMR spectra can be obtained. Four products (-243, -309, -393, and -405 ppm) have been reported. Of these complexes, the -243 and -309 ppm NMR signals apparently correspond to products containing two thiolate groups in the coordination sphere, whereas the -393 and -409 ppm products have one thiolate ligand [43], 

Although glutathione may not be quite as effective as a reducing agent, as is frequently suggested, particularly under mildly basic conditions, over time it does lead to the formation of V(IV) and the disulfide linkage of oxidized glutathione. The redox process is much more efficient under even mildly acidic conditions, where formation of V(IV) is favored. 

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It was known from quite early on (Gerbling et al., 1984) that APX was sensitive to thiol-modifying reagents (in this case 2-mercaptoethanol and dithiothreitol). In fact, inhibition by thiol reagents is one of the... 

The repair of carbohydrate radicals vithin polynucleotides can proceed with remarkable stereoselectivity, as demonstrated for hydrogen transfer from both 2-mercaptoethanol and dithiothreitol to deoxyuridin-l -yl radials within single-and double-stranded oligonucleotides (Scheme 3.3, Reactions (3.21a) and (3.21j8)) [56]. 

The sample solvent described in Section IIIA dissolves most peptides and small proteins. More effective solvents compatible with the nitrocellulose target are up to 5% (v/v) trifluoroacetic acid and up to 50% (v/v) formic acid. Urea, guanidinium chloride, and reducing agents such as mercaptoethanol and dithiothreitol are acceptable if an extensive washing procedure is applied after sample application. Fractions collected from gradient RP-HPLC containing 0.1% trifluoroacetic acid and up to 50% acetonitrile, methanol, ethanol, or 2-propanol may be applied directly without prior lyophilization provided that the sample concentration is sufficient. 

Cyclohydrolase activity in cell extract of M. kandleri is sensitive to air, but the purified enzyme is fairly air-stable [356]. Purified enzymes from M thermoautotrophicum and M. barkeri are air-stable [51,100,186]. However, the M barkeri enzyme requires nonionic detergents and ethylene glycol for stability [186]. On the other hand, cyclohydrolase activity in cell extracts of M thermoautotrophicum Marburg is stimulated 7-fold upon anaerobic incubation at 30°C in the presence of 2-mercaptoethanol or dithiothreitol, and 5-fold under the corresponding aerobic incubation [51] the mechanism of this stimulation is unknown. 

Endogenous HBV polymerase assays measure the ability of the viral polymerase to incorporate dNTPs into viral replicative intermediates present within viral core particles. If the cores are derived from secreted virions, the assay measures predominantly DDDP activity, whereas if the particles are isolated from cytosol of infected cells, the assay measures predominantly RDDP activity. In these assays, one dNTP is usually radiolabeled, and polymerase activity is detected by scintillation counting of radioactivity incorporated into acid-precipitable material, presumed to be HBV DNA. In most published assay methods, a large excess of unlabeled dNTPs is present (see Note 1). In addition to dNTP substrates, reaction mixtures used for polymerase assays typically contain a buffer (usually Tris-HCl, pH 7.5-8.0), a monovalent salt (usually KC1), essential divalent metal ions (Mg2+ or Mn2+), a reducing agent (dithiothreitol [DTT] or 2-mercaptoethanol), and a nonionic detergent (usually Nonidet-P40 or Triton X-100). Concentrations of individual components of the reaction mixture in different published assay methods vary considerably. 

Auto-oxidation of cysteine residues during cleavage of the disulfide bridge-containing proteins is a potential concern. This concern can be addressed by first reducing those proteins at alkaline pH ( 8.0) with either 2-mercaptoethanol or dithiothreitol (Equation (1)) and then alkylating with iodoacetic acid to S-carboxymethyl derivatives (Equation (2)) The reduction-alkylation process also disrupts the 3D structure of proteins to allow more sites accessible for cleavage. 

By far the most useful procedure for the reversible dissociation of apoferritin is that of Harrison and Gregory (152). Using a procedure originally derived by Fraenkel-Conrat (163) for the dissociation of viral coat proteins from their nucleic acid, it was found that treatment of apoferritin with 67% acetic acid in the cold followed by dialysis into dilute buffer at pH 3.0 generated apoferritin solvents of sedimentation coefficient 2.7 S. These subunits could be reassociated by dialysis into buffer of pH 5.0 and the extent of reassembly was found to be favoured by the addition of mercaptoethanol or dithiothreitol. An extension of these studies on the reversible dissociation of apoferritin at low pH are discussed in Section VIC. 

E. colilC. boidinii procednre twice the concentration of 1 and one-quarter the amount of NAD were nsed, and dithiothreitol was omitted. Decreasing the ratio of NAD to 1 and omitting dithio-threitol considerably decreased the materials cost of the process. Although FDH from P. pastoris is reported to be sensitive to snlfhydryl reagents, and mercaptoethanol or dithiothreitol was used during the pnrification of this enzyme as well as PDH, for a single use of the cells it was not necessary to add any dithiothreitol. 

Among the direct labeling methods, reduction of the antibody by a thiol reagent, such as mercaptoethanol or dithiothreitol, results in high labeling yields (Reilly 1993 Schwarz et al. 1988 Thakur et al. 1991). Table 2.3.2 compiles Tc-labeled antibodies approved as radiopharmaceuticals in the United States and the European Union. 

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