Mercaptoethanol, addition

Arachin, the counterpart of glycinin in peanuts, consists of subunits of 60,000—70,000 mol wt which on reduction with 2-mercaptoethanol yield polypeptides of 41,000—48,000 and 21,000 mol wt (17) analogous to the behavior of glycinin. In addition to the storage proteins, oilseeds contain a variety of minor proteins, including trypsin inhibitors, hemagglutinins, and enzymes. Examples of the last are urease and Hpoxygenase in soybeans. 

The by-products of these reactions are sulfides. The sulfide formed in the synthesis of 2-mercaptoethanol, 3-thia-l,5-pentanediol (thiodiglycol), has a variety of uses ranging from lubricant additive intermediates to textile finishing. 

Purified LBP is obtained from the crude LBP separated in the gel filtration of the 35 kDa luciferase on Sephadex G-100 (see Fig. 8.2). The fractions of crude LBP are combined and the protein is precipitated with ammonium sulfate (75% saturation). The precipitate is dissolved in a small volume of lOmM Tris-HCl/5 mM 2-mercaptoethanol, pH 8, and a small amount of luciferin is added as a tracer. Then, the crude LBP is purified on a column of Sephadex G-200 (Hastings and Dunlap, 1986). The fractions of LBP are identified by luminescence produced by the addition of luciferase at pH 6.3 the luminescence due to the tracer luciferin is proportional to the amount of LBP in each fraction. 

Depending on the choice of transfer agent, mono- or di-cnd-functional polymers may be produced. Addition-fragmentation transfer agents such as functional allyl sulfides (Scheme 7.16), benzyl ethers and macromonomers have application in this context (Section 6.2.3).212 216 The synthesis of PEG-block copolymers by making use of PEO functional allyl peroxides (and other transfer agents has been described by Businelli et al. Boutevin et al. have described the telomerization of unsaturated alcohols with mercaptoethanol or dithiols to produce telechelic diols in high yield. 

Note o-Phthaldehyde in the presence of mercaptoethanol or cysteine has already been discussed as a reagent [4]. The present monograph describes the use of o-phthal-aldehyde in the presence of sulfuric add. There are, in addition, a number of applications, which have been described, employing o-phthalaldehyde without any additives e. g. for the detection of primary arylamines, histamine, histidine and histidylpeptides [5-71. 

The aqueous ferricyanide oxidation of 2-mercaptoethanol to the disulphide is also complex kinetically" . In the pH range used (l.S. l) no complication from ionisation of the thiol is expected. Individual decays of oxidant concentrations are initially second-order but eventually become almost zero-order. For both second-and zero-order paths the rate depends on the first power of the thiol concentration and the former path is retarded by increasing the acidity, an approximately inverse relation existing above pH 3.2. Addition of ferrocyanide transforms the kinetics the rapid, second-order path is inhibited and the zero-order path is accelerated until, at 10 M ferrocyanide, the whole of the disappearance of oxidant is zero-order. Addition of Pb(C104)2, which removes product ferrocyanide, greatly enhances the oxidation rate and the consumption of oxidant becomes rs/-order. Two routes are considered to co-exist (taking due account of the acidity of ferrocyanic acid), viz. 

Wash particles (e.g., 100 mg of 1 pm carboxylated latex beads) into coupling buffer (i.e., 50 mM MES, pH 6.0 or 50 mM sodium phosphate, pH 7.2 buffers with pH values from pH 4.5 -7.5 may be used with success however, as the pH increases the reaction rate will decrease). Suspend the particles in 5 ml coupling buffer. The addition of a dilute detergent solution may be done to increase particle stability (e.g., final concentration of 0.01 percent sodium dodecyl sulfate (SDS)). Avoid the addition of any components containing carboxylates or amines (such as acetate, glycine, Tris, imidazole, etc.). Also, avoid the presence of thiols (e.g., dithiothreitol (DTT), 2-mercaptoethanol, etc.), as these will react with EDC and effectively inactivate it. 

Figure 19.19 shows a plot of the results of such an assay done to determine the maleimide content of activated BSA. This particular assay used 2-mercaptoethanol which is relatively unaffected by metal-catalyzed oxidation. For the use of cysteine or cysteine-containing peptides in the assay, however, the addition of EDTA is required to prevent disulfide formation. Without the presence of EDTA at 0.1 M, the metal contamination of some proteins (especially serum proteins such as BSA) is so great that disulfide formation proceeds preferential to maleimide coupling. Figure 19.20 shows a similar assay for maleimide-activated BSA using the more innocuous cysteine as the sulfhydryl-containing compound. 

Immediately after irradiation, stop the reaction by the addition of 7 pi of 4 X SDS electrophoresis loading buffer or the equivalent (with a high concentration of reducing agent present) 0.2M Tris, 8 percent SDS, 2.88M P-mercaptoethanol, 40 percent glycerol, 0.4 percent xylene cyanol, 0.4 percent bromophenol blue. Heat the sample at 95°C for 5 minutes and analyze the complexes formed by electrophoresis. 

The regioselectivity of Michael additions of thiolates to 2,4-dienones can be altered drastically by variation of the reaction conditions and addition of Lewis acids to the reaction mixture. Lawton and coworkers examined the reaction of 2-mercaptoethanol with l-(3-nitrophenyl)-2,4-pentadien-l-one and observed a high regioselectivity in favor of the 1,6-addition product at 45 °C (equation 42)123,124. Lowering of the reaction temperature caused an increase in the amount of 1,4-adduct, and at —40°C, a product ratio of 40 60 was found. These events suggest that kinetic control favors the 1,4-addition product whereas the 1,6-adduct is thermodynamically more stable. If, however, the reaction was carried out with a complex of the dienone and titanium tetrachloride, only the 1,4-adduct was isolated after hydrolytic workup123. Obviously, this product is trapped as a metal chelate which prevents formation of the 1,6-adduct by retro-Michael/Michael addition. In the absence of the chelating Lewis acid, the 1,4-addition product can indeed be converted... 

NDA derivatization has also been automated for analysis of amino acids in brain tissue and microdialysates (Shah et al, 1999). NDA reacts with primary amines in the presence of cyanide to form a highly stable N-substituted l-cyanobenz[/] isoindole (GBI) derivative. Addition of a nucleophile, such as cyanide, hydrogen sulphite, isothiocyanate, or 2-mercaptoethanol, is essential for the formation of the derivative. 

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