Sulfhydryl oxidation

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. 

Purify the thiolated protein from excess DTT by dialysis or gel filtration using 50 mM sodium phosphate, 0.15 M NaCl, ImM EDTA, pH 7.2. The modified protein should be used immediately in a conjugation reaction to prevent sulfhydryl oxidation and formation of disulfide crosslinks. 

The following protocol is a suggested method for labeling a protein with AMCA-HPDP. It is assumed that the presence of a sulfhydryl on the protein has been documented or created. The reaction conditions can be carried out in a variety of buffers between pH 6 and 9. Avoid the presence of extraneous sulfhydryl-containing compounds (such as disulfide reductants) that will compete in the reaction. The inclusion of EDTA in the modification buffer prevents metal-catalyzed sulfhydryl oxidation. Optimization for a particular labeling experiment should be done to obtain the best level of fluorophore incorporation. 

Dissolve the thiol-containing proteins to be crosslinked in 50 mM sodium phosphate, pH 6.5-7.5, containing 10 mM EDTA to prevent metal-catalyzed sulfhydryl oxidation. 

Add at least 5 mg/ml of a sulfhydryl-containing protein or other molecule to the SPDP-modified vesicles to effect the conjugation reaction. Molecules lacking available sulf-hydryl groups may be modified to contain them by a number of methods (Chapter 1, Section 4.1). The conjugation reaction should be done in the presence of at least lOmM EDTA to prevent metal-catalyzed sulfhydryl oxidation. 

A number of investigators have found that reagents that react with sulfhydryl groups can reproduce the symptoms of ozone damage. Dass and Weaver reported this effect in white bean. Tomlinson and Rich reported the same effect in tobacco, and Rich and Tomlinson found that conidiophores of Altemaria solani became more susceptible to ozone if they were pretreated with iodoacetamide. It appears that effects on sulfhydryl content may be quite early events in the toxic reactions initiated by ozone. One should remember, however, that symptoms of ozone and PAN injury are quite different, and one cannot explain the effects of both pollutants as sulfhydryl oxidizers. 

Finally, we conclude that toxic reactions in plant cells injured by ozone probably take place in the following sequence sulfhydryl oxidation, lipid hydrolysis, cellular leaking, lipid peroxidation, and then cellular collapse. 

Since hemoproteins such as lactoperoxidase and catalase are inhibited more rapidly than the sulfhydryl oxidation occurs, it is unlikely that the rapid activation of guanylate cyclase occurs by sulfhydryl oxidation [132]. Prolonged incubation of the papain or dehydrogenase enzymes with substrate and nitroprusside yielded a turbidity which indicated denaturation of the enzyme to an insoluble form, possibly by the formation of disulfide bridges via the dimerization of thiyl radicals [132]. 

The yield of products by the different oxidative mechanisms from peroxynitrite are strongly dependent on pH (Fig. 30). For example, the yield of expected hydroxyl radical products from dimethylsufoxide decreases with an apparent pK of approximately 7.8. The rate of sulfhydryl oxidation decreases with an apparent... 

Dissolve gelonin at a concentration of 10 mg/ml in 50 mM triethanolamine hydrochloride, pH 8, containing 10 mM EDTA. The buffer should be degassed under vacuum and bubbled with nitrogen to remove oxygen that may cause sulfhydryl oxidation after thiolation. 

Marengo, J. J., Hidalgo, C., and Bull, R. (1998). Sulfhydryl Oxidation Modifies the Calcium Dependence of Ryanodine-Sensitive Calcium Channels of Excitable Cells. Biophys J 74(3) 1263—77. 

The steady-state level of PS in the inner leaflet of plasma membrane is the result of two opposite processes, an inward translocation mediated by APT, and an outward translocation mediated by Mg2+, ATP-dependent floppase (Haest et al., 1997). Similar to APT, floppase activity could be abolished by ATP depletion and sulfhydryl oxidation (Bevers et al., 1999). 

Although xanthine oxidase does not mediate sulfhydryl oxidations per se, it is activated by the binding of hydrogen sulfide and perhaps other simple thiols (82). Thus, the effects of this enzyme also might be useful in fabricating enzymic flavor-generating systems where it could serve as an assist in limiting free thiols in the medium. 

Some conversion also results from sulfhydryl oxidation [21]. The shift of oxidase content in ischemic tissues triggers ROS formation by replenishing normal oxygen tension. Evidence for the role of xanthine oxidase as the major Oj radical source responsible for oxidative injury is the inhibition of the insult in the presence of SOD or inhibition of the enzyme activity by allopurinol. 

Trimm JL, Salama G, Abramson JJ (1986) Sulfhydryl oxidation induces rapid calcium release from sarcoplasmic reticulum vesicles. J Biol Chem 261 16092-16098... 

One electron reduction of NO generates NO. This reduction is supported by Fe(II) ion and by Fe(II)-containing metalloproteins which act as electron donors. The chemistry of NO has been less studied than NO or NO+ chemistry. However, NO is believed to mediate sulfhydryl oxidation of target proteins with the intermediate formation of RSNOH [12]. This process leads to the formation of nitrous oxide (N2O) which is also the result of NO spontaneous dimerization [12]. 

Grenier, F.C. Waygood, E.B. Saier, M.H. Bacterial phosphotransferase system regulation of the glucose and mannose enzymes II by sulfhydryl oxidation. Biochemistry, 24, 4872-4876 (1985)... 

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