Quenching with 2-mercaptoethanol

Unreacted epoxide groups are quenched with solution E (5 mL) containing mercaptoethanol (5 mM), reacting 12 h at room temperature. The devices are then... 

Similar incubations of the self-adduct in the presence of excess noncomple-mentary DNA had no effect after eight days on subsequent target alkylation. The self-adduct (1.1 pM) was even resistant to quenching by 0.5 mM 2-mercaptoethanol during a six day incubation under ambient conditions. Once again, intrastrand trapping of the transient QM seems to be extremely efficient, yet remains sufficiently dynamic for subsequently transfer of its QM to a complementary sequence (Scheme 9.22). These results are also consistent with those observed with QMP11 (Section 9.3.2). 

Brown et al.68 have developed a cellulose plate with a fluorescent indicator. Compounds are developed in 3.0% (w/v) NHfc.Cl and detected by fluorescence quenching. These authors also use 0.5% mercaptoethanol in their mobile phase, but this is only to prevent oxidation of the labile reduced pteridines, which are not adequately protected by substitution at the N5 position. Since neutral or alkaline solutions of leucovorin are relatively stable in air, this precaution may not be required for routine assay. 

Additional tests such as the addition of nucleophilic scavengers (e.g., thiols such as dithiothreitol or j8-mercaptoethanol) can provide further evidence for the presence of a free, reactive electrophilic species. The scavengers should quench all of the free reactive species, thereby protecting the enzyme from inhibition. Unfortunately, this method cannot exclude the possibility that a nucleophilic thiol may even attack the bound reactive species at the active site of the enzyme (which would also give rise to protection from inactivation). However, the use of a bulky thiol, such as reduced glutathione, should limit that possibility. An alternative scenario occurs wherein the released reactive species returns and reacts faster with an active-site nucleophile than with the added thiol. Clearly this is a complex problem and, consequently,it is advisable to use several different tests to avoid misleading conclusions. 

Figure 1. Thermal unfolding of wild type tailspike protein at 65 C. Thermal unfolding was performed by incubating 0.4 mg/ml tailspike prepared in 50 mM Tris (pH 8), 1.7 mM 2-mercaptoethanol and 2% SDS at 65°C. Samples were taken at the indicated times. The reaction was quenched by mixing the samples with SDS sample buffer (62.5 mM Tris at pH 7, 2.1 mM 2-mercaptoethanol, 10% glycerol, 0.012% Bromophenol blue dye and 2% SDS) in the cold. Then the samples were electrophoresed through SDS-PAGE at about 20°C and the proteins were stained with Coomassie blue.

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