Dithioerythritol oxidations

If the dithioerythritol oxidations do indeed follow the same mechanistic path as the mercaptoethanol oxidations with respect to relative reagent concentrations, at pH s below that observed for the maximum rate and at the high concentrations of MB , only the unimolecular disulfide radical anion formation (reaction 15) would be rate limiting. The derived rate law based on only... 

The reduction of ribonucleoside triphosphates by various dithiols which are capable of intramolecular cyclization on oxidation (dihydrolipoate, dithioerythritol, dithiothreitol) yields 2 -deoxyribonucleoside triphosphates. These reactions also require 5-deoxyadenosylcorrinoids. 

Vitamin-K-epoxide reductase (warfarin-insensitive) [EC 1.1.4.2] catalyzes the reaction of 3-hydroxy-2-methyl-3-phytyl-2,3-dihydronaphthoquinone with oxidized dithiothreitol and water to produce 2,3-epoxy-2,3-dihydro-2-methyl-3-phytyl-l,4-naphthoquinone and 1,4-dithiothreitol. In the reverse reaction, vitamin K 2,3-epoxide is reduced to 3-hydroxy- (and 2-hydroxy-) vitamin K by 1,4-dithioerythritol (which is oxidized to the disulfide). The enzyme is not inhibited by warfarin. 

Blood is collected in 2.7 ml ethylenediaminetetraacetic acid (EDTA) tubes (Sarstedt, Switzerland) containing 0.1% (w/v) dithioerythritol (DTE), immediately centrifuged at 2000 xg for 10 min, and stored at -80°C. Keep fresh or thawed plasma samples on ice during the oxidation procedure. 

Cleland (1964) showed that dithiothreitol (DTT) and dithioerythritol (DTE) are superior reagents in reducing disulfide bonds in proteins (Section 4.1). DTT and DTE have low oxidation—reduction potential and are capable of reducing protein disulfides at concentrations far below that required with 2-mercaptoethanol. However, even these reagents must be used in an approximately 20-fold molar excess in order to get close to 100% reduction of a protein. 

Fig. 7. Oxidative refolding of reduced RNase Tl. Reoxidation conditions were 0.1 M Tris-HCl, pH 7.8, 0.2 Af guanidinium chloride, 4 mM reduced glutathione, 0.4 mM oxidized glutathione, 0.2 mM EDTA, and 2.5 nM RNase Tl at 25°C. The kinetics of oxidative refolding were followed by the increase in tryptophan fluorescence intensity at 320 nm ( ), by an unfolding assay (Kiefhaber el ai, 1990b) that measures the formation of native protein molecules (A), and by the increase in the intensity of the band for native RNase Tl in native polyacrylamide gel electrophoresis ( ). Fluorescence emission in the presence of 10 mM reduced dithioerythritol to block disulfide bond formation (O). The small decrease in signal after several hours is caused by slight aggregation of the reduced and unfolded protein. (From Schonbrunner and Schmid (1992).

Biochemical reactions are often carried out with enzymes that are sensitive to oxidation. In such cases, SH reagents can be added to protect the SH groups, for example 2-mercaptoethanol at a concentration of 0.1 % (v/v) or dithiothreitol (DTT) or dithioerythritol (DTE) at a concentration of 1 mM. It should be noted that these SH reagents can be readily oxidised into compounds which absorb UV light, which can interfere with spectrophotometric measurements of concentration (see below). 

Inactivation can also be a consequence of oxidation of sulfhydryl groups, which can be avoided by the addition of mM concentrations of dithioerythritol or 2-mercap-toethanol to the buffer. Heavy metal ions can react with reactive groups on proteins, and Ca2+, Mg2+ and other divalent metal ions can promote the activity of degrading enzymes both of these effects can be suppressed by addition of mM concentrations ofEDTA. 

Physiologically, homocysteine exists in reduced, oxidized, and protein-bound forms.Methods for tHcy were first introduced in the midl980s that resolved the problems related to the presence of multiple unstable Hey species in plasma by converting all Hey species into the reduced form, HcyH, which is measured as an indication of tHcy content. Consequently, modern methods require pretreatment of plasma or serum specimens with a reducing agent, such as dithioerythritol, dithiothreitol, mercap-toethanol, tributyl phosphine, and tris(2-carboxyl-ethyl) phosphine that converts all Hey species into the reduced form, HcyH, which is measured as an indication of tHcy content. 

The effectiveness of antioxidant activity of 1, 4-dithioerythritol expressed as the radical scavenging capacity was studied by a rotational viscometry method [86]. L, 4-dithioerythritol widely accepted and used as an effective antioxidant in the field of enzyme and protein oxidation, is a new potential antioxidant standard exhibiting very good solubility in a variety of solvents. Fignre (8) describe effect of 1, 4-dithioerythri-tol on degradation of HA solntion nnder free radical stress [87]. 

It has been possible to push the reduction of a disulfide to completion by using a bisthiol, e.g., dithioerythritol (Cleland, 1964). The bisthiols owe their strong reducing power (lower redox potential) to the formation of a stable and sterically favorable dithiolane ring system upon oxidation [Eq. (2)]. 

Wheat contains glutathione and cysteine in the free state as thiol confounds (GSH, CSH), in the oxidized forms (GSSG, CSSC) and in the protein-bound forms (GSSP and CSSP) (Table 15.26). Reduction of GSSP and CSSP releases GSH and CSH respectively, e. g., with dithioerythritol. 

MB were mercaptoethanol (RSH) and dithioerythritol (R(SH)2) which were oxidized to the corresponding disulfides (RSSR and RS2, respectively) with concurrent reduction of MB to leucomethylene blue (LMB). 

The reaction rates of the oxidations of mercaptoethanol and dithioerythritol were determined at various concentration ratios of the thiols with respect to MB over a range of pH s and ionic strengths in both water and D O by following the decrease in the... 

MB Oxidations of Dithioerythritol. Although the kinetic rate laws observed for the mercaptoethanol oxidations are... 

As in the case of the mercaptoethanol oxidations, the oxidation rates of dithioerythritol are pH-dependent showing a maximum at pH 9.6 (see Table IV). The lower pH observed for the maximum rate relative to that observed for mercaptoethanol (see Table I) parallels the lower pK for the first ionization... 

The observed rate laws for the methylene blue oxidations of both mercaptoethanol and dithioerythritol at the different pH s are consistent with the mechanisms proposed for these oxidations. The most significant effect of the pH on the reaction is that of establishing the relative amounts of thiol and sulfide ion. Initiation of the chain sequence by the reaction of a sulfide ion with MB+ would be expected to be more rapid, and therefore the overall oxidation. faster, if the pH of the medium is increased. However, at the higher pH s where the relative amount of undissociated thiol is small, the reaction rate diminishes indicating not only that the thiol is likely a reactant in the overall reaction but that it is involved in a rate limiting step of the reaction at higher pH s. While not as supportive as the... 

Solvent Isotope Effects. The involvement of the abstraction of the hydrogen atom from the thiol by MB- as a rate limiting factor at the higher pH s is supported by comparison of the oxidation reaction rates in H2O and D2O at different pH s. Table VII lists the observed rate constants for the oxidations of both mercaptoethanol and dithioerythritol at different acidities. 

The effects of pH, ionic strength, reagent concentration ratios and deuterium substitution of the sulfur-bonded hydrogens on both the rates and rate laws for the methylene blue oxidations of mercaptoethanol and dithioerythritol have been determined. A free radical chain mechanism consistent with the observed kinetic behavior of the oxidation reactions is proposed. A key feature of the proposed mechanism is the formation of the sulfur-sulfur linkage of the disulfide in the reversible formation of a disulfide radical anion (RSSR) as a chain propagating step in the chain sequence. 

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