Oxidation of sulfhydryls

Dissolve the sulfhydryl-containing protein or macromolecule to be modified at a concentration of l-10mg/ml in 50mM Tris, 0.15M NaCl, 5mM EDTA, pH 8.5. EDTA is present to prevent metal-catalyzed oxidation of sulfhydryl groups. The presence of Tris, an amine-containing buffer, should not affect the efficiency of sulfhydryl modification. Not only do amines generally react slower than sulfhydryls, the amine in Tris buffer is of particularly low reactivity. If Tris does pose a problem, however, use 0.1M sodium phosphate, 0.15M NaCl, 5mM EDTA, pH 8.0. 

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.

Chang has made observations on the polysomes of pinto bean leaves exposed to ozone (at 0.35 ppm for 20-50 min). He found that the chloroplast polysomes were more susceptible to oxidation than was the cytoplasmic ribosomes. The sulfhydryl content of the chloroplast ribosomes was also much more susceptible to oxidation than was that of the cytoplasmic ribosomes. Finally, it was found that the effects of ozone on ribosome composition could be reproduced by p-mer-curicbenzoate. Chang s results imply that either ozone itself or a product of ozone oxidation passes from the cytoplasmic membrane to the interior of the chloroplast before having its effect. These results connect with a number of papers on the oxidation of sulfhydryl compounds by ozone. Tomlinson and Rich have reported decreases in leaf sulfhydryl groups after ozone exposure (at 1 ppm for 30-60 min). 

Radi, R., Beckman, J. S., Bush, K. M., and Freeman, B. A. (1991). Peroxynitrite oxidation of sulfhydryls The cytotoxic potential of superoxide and nitric oxide. J. Biol. Chem. 266, 4244-4250. 

It is usually believed that NO inhibits enzymes by reacting with heme or nonheme iron or copper or via the S-nitrosilation or oxidation of sulfhydryl groups, although precise mechanisms are not always evident. By the use of ESR spectroscopy, Ichimori et al. [76] has showed that NO reacts with the sulfur atom coordinated to the xanthine oxidase molybdenum center, converting xanthine oxidase into a desulfo-type enzyme. Similarly, Sommer et al. [79] proposed that nitric oxide and superoxide inhibited calcineurin, one of the major serine and threonine phosphatases, by oxidation of metal ions or thiols. 

Dissolve the antibody to be modified at a concentration of 1—10 mg/ml in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2, containing 10 mMEDTA. High levels of EDTA often are required to completely stop metal-catalyzed oxidation of sulfhydryl groups when working with serum proteins—especially polyclonal antibodies purified from antisera. Presumably, carryover of iron from partially hemolyzed blood is the contaminating culprit. 

Further results from these Raman studies showed that the lens nucleus and cortex had somewhat different amino acid compositions. It could be concluded that the nuclear portion has highest concentration of y-crystallin, and that the content of a-crystallin increases significantly from the nucleus to the cortex. Furthermore, it was found that sulfhydryl groups and (i-conformation are unaffected in the conversion from a transparent to totally opaque lens by heat denaturation. This indicates that the opacification of the lens does not necessarily involve the oxidation of sulfhydryl groups or conformational changes [3],... 

Post-translational modifications were recognized as additional sources of the structural modification of proteins.(22) Should such a modification occur by an enzyme-mediated process, as had been established for oxidation of sulfhydryl groups or the addition of carbohydrate or phosphate groups, or by the cleavage of the polypeptide with loss of a terminal amino group, or a larger part of the chain, it too could be subject to genetic variation. 

Often it is necessary to prevent the oxidation of sulfhydryl groups, as the presence of disulfide links could lead to insolubility of a protein or to inability to determine its sequence. It is possible to block the reactive —SH groups with a range of chemical reagents. 

Many, but not all, proteins are sensitive to alterations in the oxidation-reduction potential of their environment. The effect is caused in part by oxidation of sulfhydryl groups or reduction of disulfide bonds. Not all proteins are equally sensitive to such alterations, but when they are, it is critical to be aware of their sensitivity. The purification or assay of some proteins can be accomplished only by providing reducing conditions (reduced glutathione, free cysteine, dithiothreitol, or mercap-toethanol) in all buffer solutions. 

Oxidation of sulfhydryl groups leads to formation of intra- and intermolecular disulfide bridges but may also go further, with formation of sulfoxides and sulfones... 

The biological function of many of these posttranslational modification reactions is also tenuous at this stage, even if we can rationalize some of them as components of well understood processes. The most obvious example of such established processes is the oxidation of sulfhydryl groups resulting in the formation of disulfide bridges which are essential structural... 

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. 

Chloramine-T radioiodination of proteins involves both oxidation of radioiodide to its reactive state, H2OD, and oxidation of sulfhydryl groups of the protein molecules. There must be sufficient chloramine-T... 

Radi R, Beckman JS, Bush BCM, Freeman BA (1991) Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitiic oxide. J Biol Chem 266 4244 250. 

The S-S linkages are formed by the oxidation of sulfhydryl (-SH) group of two cysteine side chains. 

The awareness of the many advantages of this modification reaction should be matched by the knowledge of the numerous side-reactions observed to result from the reaction of TNM with proteins. In addition to nitration of tyrosine, the following side-reactions have been reported in several (but not all) proteins studied (1) inter- and intramolecular cross-linking, (2) oxidation of sulfhydryl groups to a variety of products, (3) oxidation of methionine, (4) modification of tryptophan and histidine, (5) modification of prosthetic groups. It is apparent therefore that a successful application of TNM to the selective modification of tyrosine is achieved in those cases where an unusually rapid reaction... 

Oxidations of sulfhydryl groups to disulfides, of alcohols to aldehydes and ketones, and of aldehydes to carboxylic acids frequently occur. 

Research on the biochemical effects of 03 has been extensive. Among the many mechanistic postulations that have been advanced concerning the toxicity of 03, the following are noted (1) reactions with proteins and amino acids (2) reactions with lipids (3) formation of free radicals (4) oxidation of sulfhydryl compounds and pyridine nucleotides (5) influence on various enzymes and (6) production of more or less nonspecific stress, with the release of histamine. 

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