Disulfide formation

NO2C6H4SCI, AcOH (results in disulfide formation), followed by NaBH4 or HS(CH2)20H or dithioerythritol, quant." 5-Triphenylmethyl, 5-4,4 -di-methoxydiphenylmethyl, and 5-acetamidomethyl groups are also removed by this method. 

II. Thiol oxidants cystaminc (mixed disulfide formation), diamide, t-BHP, menadione, diquat... 

This thiol-disulfide interconversion is a key part of numerous biological processes. WeTJ see in Chapter 26, for instance, that disulfide formation is involved in defining the structure and three-dimensional conformations of proteins, where disulfide "bridges" often form cross-links between q steine amino acid units in the protein chains. Disulfide formation is also involved in the process by which cells protect themselves from oxidative degradation. A cellular component called glutathione removes potentially harmful oxidants and is itself oxidized to glutathione disulfide in the process. Reduction back to the thiol requires the coenzyme flavin adenine dinucleotide (reduced), abbreviated FADH2. 

Mann JR, Watson DP (2007) Adsorption of CdSe nanoparticles to thiolated TiOa surfaces Influence of intralayer disulfide formation on CdSe surface coverage. Langmuir 23 10924-10928... 

Jaeschke, H. (1990). Glutathione disulfide formation and oxidant stress during acetaminophen-induced hepatotoxicity in mice in vivo-, the protective effect of allopurinol. J. Pharmacol Exp. Ther. 255, 935-941. 

Kamura T Tsuda H., Yae Y., et al. An abnormal fibrinogen Fukuoka II (Gly-B(315- CY5) characterized by defective fibrin lateral association and mixed disulfide formation. J Biol Chem 1995 270,29392-9. 

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 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... 

The deacetylated protein should be used immediately to prevent loss of sulfhydryl content through disulfide formation. The degree of—SH modification may be determined by performing an Ellman s assay (Section 4.1, this chapter). 

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. 

In an attempt to sensitize the thiosulfate bond cleavage, benzophenone (10% by weight) was incorporated into the polymer film. Upon photolysis at 366 nm, the 639 cm 1 thiosulfate band was reduced (Figure 10) as in the case of direct photolysis at 254 nm and 280 nm. Since benzophenone is a known triplet sensitizer it is likely that the S-S bond cleavage in the thiosulfate group occurs from a triplet excited state in the sensitized reaction. Incidentally photolysis of a PATE film at 366 nm in the absence of benzophenone resulted in no loss of the 639 cm 1 IR peak. Unfortunately due to the film thickness, we were unable to obtain accurate quantum yields for either the direct or sensitized photolysis. Finally it should be noted that no chemical evidence has been presented to confirm disulfide formation. Results from the photolysis of a PATE-type model compound will be offered to substantiate the claim of disulfide formation as well as quantitate the primary photolysis step. But first, we consider photolysis of a PASE polymer film. 

Figure 4.2 Disulfide formation between two cysteine residues. The product of the oxidation reaction, stable to acid hydrolysis is called cystine.

DS Kemp, S-L Leung, DJ Kerkman. Models that demonstrate peptide bond formation by prior thiol capture. 1. Capture by disulfide formation. Tetrahedron Lett 22, 181, 1981. 

D. R. Arnelle, J. S. Stamler, NO+, NO, and NO Donation by 5-Nitrosothiols Implications for Regulation of Physiological Functions by 5-Nitroxylation and Acceleration of Disulfide Formation , Arch. Biochem. Biophys. 1995, 318, 279-285. 

N. F. Tabachnik, P. Blackburn, C. M. Peterson, A. Cerami, Protein Binding of JV-2-Mer-captoethyl-2,3-diaminopropane via Mixed Disulfide Formation after Oral Administration of WR 2721 , J. Pharmacol. Exp. Ther. 1982, 220, 243 - 246. 

The kinetics of disulfide formation, the demonstration of specific binding, and the immunochemical results all support the conclusion that native-like structure results from the oxidative folding of reduced peptide 13-105. These three independent lines of evidence support the conclusion that lysozyme has a continuous chain independent assembly region somewhere in the sequence 13-105. 

Disulfide bridges are, of course, true covalent bonds (between the sulfurs of two cysteine side chains) and are thus considered part of the primary structure of a protein by most definitions. Experimentally they also belong there, since they can be determined as part of, or an extension of, an amino acid sequence determination. However, proteins normally can fold up correctly without or before disulfide formation, and those SS links appear to influence the structure more in the manner of secondary-structural elements, by providing local specificity and stabilization. Therefore, it seems appropriate to consider them here along with the other basic elements making up three-dimensional protein structure. 

The most common posttranslational modifications, discussed in the following sections, include phosphorylation, sulfation, disulfide formation, N-methylation, O-methylation, S-methylation, N-acetylation, hydroxylation, glycosylation, ADP-ribosylation, prenylation, biotinylation, lipoylation, and phosphopan-tetheine tethering. Many of the posttranslational modifications are proven to be cross talks. Other modifications exist in a smaller extent and include oxidation of methionine, C-methylation, ubiquitylation, carboxylation, and amidation. These topics will not be covered in this chapter which is meant to focus primarily on the recent literature (2005-08). For a more complete coverage of all posttranslational modifications and earlier literature (up to 2005), the reader is referred to Professor Christopher T. Walsh s book Posttranslational Modification of Proteins Expanding Nature s Inventory ... 

Posttranslational modifications can be broken down into two main classes those that are reversible and those that are irreversible. Included in the large group of reversible posttranslational modifications are phosphorylation, acetylation, and disulfide formation. Irreversible posttranslational modifications include peptide bond cleavage as in intein splicing also irreversible is the introduction of a phosphopantetheinyl group during fatty acid, polyketide, and nonribosomal peptide biosyntheses. The current debate is whether to classify lysine N-methylation as reversible or irreversible. Recently, there have been reports of lysine demethylases. ... 

Cysteine disulfide formation is one of the most important posttranslational modifications involved in protein structure. Disulfides play a crucial role in maintaining the structure of many proteins including insulin, keratin, and many other structurally important proteins. While the cytoplasm and nucleus are reducing microenvironments, the Golgi and other organelles can have oxidizing environments and process proteins to contain disulfide bonds (Scheme 5). 

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