2-Mercaptoethanol, oxidation

Fig. 20. Influence of DDS concentration on the starting rate of 2-mercaptoethanol oxidation (1) and the turbidity of the system (2) [147, 156]...

Fig. 9.4 The enzyme RNase can be unfolded by reduction agents (urea and mercaptoethanol). Oxidative removal of the reduction agents causes the molecule to take up its three-dimensional structure again it regains almost its full enzymatic activity...

It was determined that the molecular weight of a polymer ligand greatly affects the rate of mercaptoethanol oxidation [151-153]. Fig. 18 presents the dependence of 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... 

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... 

Effect on Ionic Strength on Mercaptoethanol Oxidation Rates Rate X 10 M sec ... 

Mercaptoethanol Mesityl oxide a-Methacrylic acid Methane... 

FIGURE 5.18 Methods for cleavage of disulfide bonds in proteins, (a) Oxidative cleavage by reaction with performic acid, (b) Reductive cleavage with snlfliydryl compounds. Disulfide bridges can be broken by reduction of the S—S link with snlfliydryl agents such as 2-mercaptoethanol or dithiothreitol. Because reaction between the newly reduced —SH groups to re-establish disulfide bonds is a likelihood, S—S reduction must be followed by —SH modification (1) alkylation with iodoac-etate (ICH,COOH) or (2) modification with 3-bromopropylamine (Br— (CH,)3—NH,). 

Ribosomal protein L12 was oxidized with 0.3 M H202 at 30°C for 1 h. After dialysis, the protein was incubated in the presence of 0.8 M 2-mercaptoethanol for 48 min at 37 °C and dialyzed. The amount of methionine residues was quantitated by exhaustive alkylation of the protein with [14C]iodoacetic acid. 

Ribosomal protein L12 was oxidized with N-chlorosuccinimide as described by Schechter and coworkers28 and dialyzed. The complete system contained 33 mM Tris-HCI (pH 7.4), 13mM MgCI2,275 pmol Met(0)-L12,13mM dithiothreitol (or 2-mercaptoethanol where indicated), and enzyme. See the legend to Figure 5 for further details of the assay. 

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. 

Cleland (1964) showed that DTT and DTE are superior reagents in reducing disulfide bonds in proteins (see previous discussion, this section). 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 have to be used in approximately 20-fold molar excess in order to get close to 100 percent reduction of a protein. 

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. 

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. 

Shinkai and Kunitake (1977b) prepared a hydrophobic flavin analogue, 3-hexadecyl-10-butylisoalloxazine [56]. Its absorption spectrum in CTAB micelles showed distinct shoulders at 420 nm and 460-470 nm, as in the flavin spectrum in organic solvents. This indicates that [56] is located in the hydrophobic region of the micelle. Isoalloxazine [56] bound to a cationic micelle readily oxidizes 2-mercaptoethanol, 1,4-butanedithiol, and thiophenol (Shinkai and Kunitake, 1977b Shinkai et al., 1977a). In non-micellar... 

Primary amino acids will react with o-phthalaldehyde in the presence of the strongly reducing 2-mercaptoethanol (pH 9-11) to yield a fluorescent product (emission maximum, 455 nm excitation maximum, 340 nm). Peptides are less reactive than a-amino acids and secondary amines do not react at all. As a result, proline and hydroxyproline must first be treated with a suitable oxidizing agent such as chloramine T (sodium A-chloro-p-toluene-sulphonamide) or sodium hypochlorite, to convert them into compounds which will react. Similarly cystine and cysteine should also be first oxidized to cysteic acid. 

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