Mercaptoethanol disulfide

A fermented-egg product (EEP), patented as an attractive bait for synanthropic flies, has been shown to be attractive to coyotes and repeUent to deer (79). Its components are variable, with relative concentrations of 77% fatty acids, 13% bases, and 10% (primarily) neutrals composed of at least 54 volatiles such as ethyl esters, dimethyl disulfide, and 2-mercaptoethanol. Synthetic formulations have been evaluated to find a replacement for a patented fermented-egg protein product that attracts coyotes and repels deer. Ten aUphatic acids (C-2 to C-8), four amines (pentyl, hexyl, heptyl, and trimethyl), dimethyl disulfide, 2-mercaptoethanol, and 54 more volatiles (C-1 to C-5 esters of C-1 to C-8 acids) have been tested as synthetic fermented egg (SEE) (80) in approximately the same proportions that are present in EEP. Weathering was a problem that caused decreased efficacy, which suggests trials of controUed-release formulations. Eourteen repeUents have been examined against white-taU deer in Peimsylvania in choice tests when treated onto sheUed com (81). 

Fig. 3. Sodium dodecyl sulfate—polyacrylamide gel electrophoretic pattern for molecular weight standards (lane 1) water-extractable proteins of defatted soybean meal (lane 2) purified IIS (glycinin) (lane 3) and purified 7S (P-conglycinin) (lane 4) where the numbers represent mol wt x 10. The gel was mn in the presence of 2-mercaptoethanol, resulting in the cleavage of the disulfide bond linking the acidic (A bands) and basic (B bands) polypeptides of the...

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,). 

Penicillin G. acylase, pH 7.8 buffer, 35°, 30 min to 2 h. These conditions result in isolation of the disulfide, but if j8-mercaptoethanol is included in the reaction mixture, the thiol can be isolated. ... 

Tissue surrogates heated in 6M guanidine HC1 supplemented with 0.5 M P-mercaptoethanol (BME), a disulfide-reducing agent, resulted in a protein... 

Dithiothreitol (DTT) and dithioerythritol (DTE) are the trans and cis isomers of the compound 2,3-dihydroxy-1,4-dithiolbutane. The reducing potential of these versatile reagents was first described by Cleland in 1964. Due to their low redox potential (—0.33 V) they are able to reduce virtually all accessible biological disulfides and maintain free thiols in solution despite the presence of oxygen. The compounds are fully water-soluble with very little of the offensive odor of the 2-mercaptoethanol they were meant to replace. Since Cleland s original report, literally thousands of references have cited the use of mainly DTT for the reduction of cystine and other forms of disulfides. 

The reduction of disulfides by 2-mercaptoethanol proceeds through a mixed disulfide intermediate. 

Many extracellular proteins like immunoglobulins, protein hormones, serum albumin, pepsin, trypsin, ribonuclease, and others contain one or more indigenous disulfide bonds. For functional and structural studies of proteins, it is often necessary to cleave these disulfide bridges. Disulfide bonds in proteins are commonly reduced with small, soluble mercaptans, such as DTT, TCEP, 2-mercaptoethanol, thioglycolic acid, cysteine, etc. High concentrations of mercaptans (molar excess of 20- to 1,000-fold) are usually required to drive the reduction to completion. 

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. 

Inhibition of the dinuclear Ni(II) enzyme urease by Bi(III) thiolates may be important for its antibacterial activity. Helicobacter pylori relies on urease for the production of ammonia which allows survival under the highly acidic conditions of the gastric lumen and mucosa. Bismuth(III) mercaptoethanol complexes are ca. 103 more active inhibitors than mercaptoethanol alone (466). The thiol can bind directly to Ni(II) in the active site and also form a disulfide with a Cys residue in the active site cavity (467). 

Figure 2.12 MS data of tethered complexes, (a) Tethering experiment between TS and 10 disulfides equilibrated for 1 hour, (b) tethering with varying concentrations of 2-mercaptoethanol, and (c) tethering with varying pool size of disulfides. Reproduced from Reference 31 with permission of the National Academy of Sciences, USA. Copyright (2000) National Academy of Sciences, U.S.A.

The disulfide bonds can be reductively cleaved by thiols (e.g., mercaptoethanol, HO-CH2-CH2-SH). If urea at a high concentration is also added, the protein unfolds completely. In this form (left), it is up to 35 nm long. Polar (green) and apolar (yellow) side chains are distributed randomly. The denatured enzyme is completely inactive, because the catalytically important amino acids (pink) are too far away from each other to be able to interact with each other and with the substrate. 

---