Preventing oxidation disulfides

Blocking of unreacted thiol groups with /V-ethylmaleimide prevents oxidative disulfide-bond formation between surface thiols and cysteine residues of proteins during incubation with labeled proteins. 

Purify the thiolated protein from unreacted Traut s reagent by gel filtration using your buffer of choice (i.e., 20mM sodium phosphate, 0.15M NaCl, ImM EDTA, pH 7.2). The addition of EDTA to this buffer helps to prevent oxidation of the sulfhydryl groups and the resultant disulfide formation. After purification, use the thiolated protein immediately... 

It is necessary to fractionate as rapidly as possible in order to prevent oxidation to the disulfide, which occurs almost completely even in the presence of nitrogen if the column is too long or if the distillation is carried out too slowly. Oil-pumped nitrogen is dried through an absorption tower containing soda lime and calcium chloride before passing to the distillation apparatus. The column should be vacuum jacketed or provided with a heated jacket. 

The aim is to extract protein molecules as pure as possible. Detergents generally help membrane proteins to dissolve and separate from lipids. Reductants are used to reduce disulfide bonds or prevent oxidation. Denaturing agents alter ionic strength... 

M sodium phosphate, 0.15 M NaCl, pH 7.5, containing 10 mM EDTA. The presence of EDTA in this buffer helps to prevent oxidation of the sulfhydryl groups with resultant disulfide formation. The degree of—SH modification in the purified protein may be determined using the Ellman s assay (Chapter 1, Section 4.1). 

While the hydrogen sulfide formed during fermentation can be easily removed with the addition of copper sulfate, the international legal limit of 0.2 mg/L residue in wines and the natural complexing and binding of copper ions in wines often prevent suitable additions from being made in practice. Of more concern is the secondary thiols and thioacetates that are produced with hydrogen sulfide (J) and the inability of copper to remove them or their oxidized disulfide forms. 

Oxidizing conditions result in formation of disulfide bonds and intermolecular interactions are expected. Reducing conditions will prevent intermolecular disulfide bond formation. 

The first step in determining the sequence of amino acids in a peptide or a protein is to reduce any disulfide bridges in the peptide or protein. A commonly used reducing agent is 2-mercaptoethanol, which is oxidized to a disulfide. Reaction of the protein thiol groups with iodoacetic acid prevents the disulfide bridges from reforming as a result of oxidation by O2. 

The formation of protein-glutathione mixed disulfide prevents oxidation of protein SH groups to, for example, a sulfonic acid derivative (PSO3). The GS- and PS radicals derived from the action of r-butyl peroxide (r-BuOOH) on GSH and PSH (equations 5,6) can dimerize to form PS-SG (equation 7). The net effect is protection by glutathione of sensitive protein SH groups against oxidative damage. 

Note that we used thioacetate (sometimes referred to as thiolacetate) end groups since these could be selectively deprotected in solution, to afford the free thiol, using NH4OH or acid during the deposition process. " Alkali metal salts can be avoided since they tend to disrupt electronics measurements. Use of the free thiols, rather than the thioacetates, proved to be somewhat problematic since they were prone to very rapid oxidative disulfide formation. But if a quality inert atmosphere box is used, use of the free thiol can be effective a N2-flush bag is not adequate to prevent the aromatic disulfide formation. Furthermore, the disulfides formed can self-assemble on gold, but the assembly is approximately KXX) times slower than with the thiols. When using the a,(i)-dithiols, oxidative polymerization ensues which rapidly results in insoluble material. Hence, in situ removal of the acetates has proven to be quite effective although not essential if strict exclusion of air is maintained. 

Proteins modified with 2-iminothiolane are subject to disulfide formation upon sulfhydryl oxidation. This can cause unwanted conjugation, potentially precipitating the protein. The addition of a metal-chelating agent such as EDTA (0.01-0.1M) will prevent metal-catalyzed oxidation and maintain sulfhydryl stability. In the presence of some serum proteins (i.e., BSA) a 0.1M concentration of EDTA may be necessary to prevent metal-catalyzed oxidation, presumably due to the high contamination of iron from hemolyzed blood. 

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