Cysteine preventing oxidation

Moriarty-Craige, S. Adkison, J. Lyim, M. Gensler, G. Bressler, S. Jones, D. Stemberger, P. 2005. Antioxidant supplements prevent oxidation of cysteine/ cystine redox inpatients with age-related macular degeneration. Am. J. Ophthalmol. 140 1020-1026. 

There is no known antidote to paraquat or diquat poisoning. Some clinicians have used antioxidants such as vitamins C or E, N-acetyl cysteine, nitric oxide donors, and a combination of cyclophosphamide and corticosteroids to prevent inflammation and pulmonary fibrosis in severe cases. However, the effectiveness of these treatments appears to be marginal. If the chemical has been ingested, the main treatment is to prevent further absorption from the gastrointestinal tract. This is accomplished by the standard methods gastric lavage with safine solution to wash the substance from the stomach or ingestion of activated charcoal or Fuller s earth to act as absorbents. Since one of the effects of both paraquat and... 

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. 

In some instances, it might be necessary to supplement the 0.32M sucrose solution, pH 7.4, with 1 mAf EDTA, 0.25 mM dithiothreitol (DTT), and/or a cocktail of protease inhibitors. Membrane-bound phospholipases and proteases can be activated during cell disruption. EDTA chelates metal ions (calcium and magnesium) that activate certain phospholipases. DTT is a reducing agent that prevents oxidation of functionally important sulfhydryl groups. Cell disruption may also cause release of proteases from lysosomes. Protease attack on membrane proteins can be prevented by addition of protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF an inhibitor of serine proteases) and/or E-64 (an inhibitor of cysteine proteases), to the sucrose solution... 

Joshi, G., S. Hardas, R. Sultana et al. Glutathione elevation by ganuna-glutamyl cysteine ethyl ester as a potential therapeutic strategy for preventing oxidative stress in brain mediated by in vivo administration of adriamycin Implication for chemobrain. 

Another advantage to the use of a thiol additive is that the abundance of free thiol groups in the reaction environment will prevent the oxidation of the cysteine thiol at the N-terminal of the other peptide. Without added thiol transesterification catalysts, disulfide formation resulting in dimerization of the Cys-peptide would be a dominant side reaction in aqueous, oxygenated buffer conditions. 

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. 

Figure 19.20 Cysteine also may be used in an Ellman s assay to determine the maleimide activation level of SMCC-derivatized proteins. Reaction of the activated carrier with different amounts of cysteine results in various levels of sulfhydryls remaining after the reaction. The coupling must be done in the presence of EDTA to prevent metal-catalyzed oxidation of sulfhydryls. Detection of the remaining thiols using an Ellman s assay indirectly indicates the amount of sulfhydryl uptake into the activated carrier. Comparison of the Ellman s response to the same quantity of cysteine plus an unactivated carrier indicates the absolute amount of sulfhydryl that reacted. Calculation of the maleimide activation level then can be done.

The principle of oxidation of bis-cysteine peptides on resin consists of deprotection and oxidation of a cysteine pair in a single step or in two separate steps to form the disulfide bond while the peptide chain remains attached to the solid support. The strategy is especially effective for the formation of intramolecular disulfides since the solid support simulates high dilution via the pseudo-dilution effect, thus, largely preventing intermolecular reactions. 163 98 10° ... 

The general strategy is based on selective deprotection and oxidation of pairwise cysteine residues, as specified by the orthogonal protection scheme selected for this purpose. Since multiple disulfide bonds are formed step-by-step, reaction conditions are required that prevent breaking or scrambling of the disulfide bonds already formed. Therefore, throughout the synthesis, exposure to thiols or alkaline conditions as well as lengthy reaction times that may cause disulfide disproportionation must be avoided. 

The inherent drawbacks of the oxidative refolding approach for synthetic polypeptides containing multiple cysteine residues is the individual behavior of each peptide that derives from the encoded sequence, more or less pronounced structural information which prevents general procedures to be elaborated and proposed. Nevertheless, this synthetic approach remains attractive because of its simplicity compared to the synthetic strategies for re-gioselective disulfide bond formation (Section 6.1.1-6.1.4), and it is certainly indispensable if the number of cysteine residues exceeds the presently available chemistry for site-directed cysteine pairings. 

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