Disulfide reductants reactions

N204 also forms expl mixts with incompletely halogenated hydrocarbons, NGu, carbon disulfide, etc (Ref 33). The effect of spontaneous decompn by oxidation-reduction reactions when N204 is mixed with a number of fuels (hydrazine, gasoline, liq paraffin, etc) has resulted in its extensive use in liq propint rocket engines (Refs 12, 22, 27 35)... 

Disulfide reduction occurs over a broad pH range and in a variety of buffer environments. The reaction can be done in denaturants, chaotropic agents, detergents, and in high salt conditions. 

The following protocol for labeling proteins with 5-IAF is adapted from Gorman (1987). It is a bit unusual in that it involves reduction of disulfides with dithiothreitol (DTT) and immediate reaction with 5-IAF in excess without removal of excess reductant. The procedure can be changed to include a gel filtration step after disulfide reduction to remove excess DTT, but in any case, it should be optimized for each protein to be modified. An alternative to the use of DTT to produce sulfhydryls is thiolation with a compound that can generate free thiols upon reaction with a protein (Chapter 1, Section 4.1). 

For complete reduction of all disulfides in the presence of a denaturant, react for 16 hours at 0°C and 2 hours at room temperature. For partial reduction of disulfides, the reaction time may be reduced to 2 hours at 37°C, particularly for antibody thiol reduction, if only partial reduction of thiols in the hinge region is done. 

The following protocol is a suggested method for labeling a protein with AMCA-HPDP. It is assumed that the presence of a sulfhydryl on the protein has been documented or created. The reaction conditions can be carried out in a variety of buffers between pH 6 and 9. Avoid the presence of extraneous sulfhydryl-containing compounds (such as disulfide reductants) that will compete in the reaction. The inclusion of EDTA in the modification buffer prevents metal-catalyzed sulfhydryl oxidation. Optimization for a particular labeling experiment should be done to obtain the best level of fluorophore incorporation. 

Traditional sample preparation conditions to form SDS-protein complexes prior to electrophoretic analysis included heat treatment at elevated temperatures (e.g., In the case of non-reduced rMAbs, this could lead to sample preparation artifacts in the form of thermally induced fragmentation attributed to disulfide reduction and exchange reactions. Moreover, it was reported that high pH (>9.0) also enhanced the... 

To further exploit the potential usefiilness of this new family of clusters, monoadduct 54 was saponified into 55 (0.05 M NaOH, quant) and condensed to L-lysine methyl ester using 2-ethoxy-l-ethoxycarbonyl-l,2-dihydroquinoline (EEDQ) to give extended dimer 56 in 50 % yield together with monoadduct in 15 % yield [75]. Additionally, tert-butyl thioethers 52 could be transformed into thiols by a two step process involving 2-nitrobenzenesulfenyl chloride (2-N02-PhSCl, HOAc, r.t, 3h, 84%) followed by disulfide reduction with 2-mercaptoethanol (60%). Curiously, attempts to directly obtain these thiolated telomers by reaction with thioacetic acid f ed. These telomers were slightly better ligands then lactose in inhibition of binding of peanut lectin to a polymeric lactoside [76]. 

The fifth cofactor of the PDH complex, lipoate (Fig. 16-4), has two thiol groups that can undergo reversible oxidation to a disulfide bond (—S—S—), similar to that between two Cys residues in a protein. Because of its capacity to undergo oxidation-reduction reactions, lipoate can serve both as an electron hydrogen carrier and as an acyl carrier, as we shall see. 

In the first step of the conversion catalyzed by pyruvate decarboxylase, a carbon atom from thiamine pyrophosphate adds to the carbonyl carbon of pyruvate. Decarboxylation produces the key reactive intermediate, hydroxyethyl thiamine pyrophosphate (HETPP). As shown in figure 13.5, the ionized ylid form of HETPP is resonance-stabilized by the existence of a form without charge separation. The next enzyme, dihydrolipoyltransacetylase, catalyzes the transfer of the two-carbon moiety to lipoic acid. A nucleophilic attack by HETPP on the sulfur atom attached to carbon 8 of oxidized lipoic acid displaces the electrons of the disulfide bond to the sulfur atom attached to carbon 6. The sulfur then picks up a proton from the environment as shown in figure 13.5. This simple displacement reaction is also an oxidation-reduction reaction, in which the attacking carbon atom is oxidized from the aldehyde level in HETPP to the carboxyl level in the lipoic acid derivative. The oxidized (disulfide) form of lipoic acid is converted to the reduced (mer-capto) form. The fact that the two-carbon moiety has become an acyl group is shown more clearly after dissocia-... 

The unique characteristics of DTT and DTE are mainly reflected in their ability to form intramolecular ring structures upon oxidation. Disulfide reductants such as 2-mercaptoethanol, 2-mercaptoethylamine, glutathione, thioglycolate, and 2,3-dimercaptopropanol cleave disulfide bonds in a two-step reaction that involves the II formation of a mixed disulfide (Fig. 66). In the second stage of the reducing process, the... 

Grollmann U, Schnabel W (1980) On the kinetics of polymer degradation in solution, 9. Pulse radiolysis of polyethylene oxide). Makromol Chem 181 1215-1226 Hamer DH (1986) Metallothionein. In Richardson CC, Boyer PD, Dawid IB, Meister A (eds) Annual review of biochemistry. Annual Reviews, Palo Alto, pp 913-951 Held KD, Harrop HA, Michael BD (1985) Pulse radiolysis studies of the interactions of the sulfhydryl compound dithiothreitol and sugars. Radiat Res 103 171-185 Hilborn JW, PincockJA (1991) Rates of decarboxylation of acyloxy radicals formed in the photocleavage of substituted 1-naphthylmethyl alkanoates. J Am Chem Soc 113 2683-2686 Hiller K-O, Asmus K-D (1983) Formation and reduction reactions of a-amino radicals derived from methionine and its derivatives in aqueous solutions. J Phys Chem 87 3682-3688 Hiller K-O, Masloch B, Gobi M, Asmus K-D (1981) Mechanism of the OH radical induced oxidation of methionine in aqueous solution. J Am Chem Soc 103 2734-2743 Hoffman MZ, Hayon E (1972) One-electron reduction of the disulfide linkage in aqueous solution. Formation, protonation and decay kinetics of the RSSR radical. J Am Chem Soc 94 7950-7957... 

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