Cystine, disulfide reduction

U. T. Ruf gg u. J. Rudinger, Reductive Cleavage of cystine disulfides with tributylphosphins, Methods Enzymol. 47, 111 (1977). 

Cysteine sulfhydryls and cystine disulfides may undergo a variety of reactions, including alkylation to form stable thioether derivatives, acylation to form relatively unstable thioesters, and a number of oxidation and reduction processes (Figure 1.10). Derivatization of the side chain sulfhydryl of cysteine is one of the most important reactions of modification and conjugation techniques for proteins. 

One of the most convenient ways of generating sulfhydryl groups is by reduction of indigenous disulfides. Many proteins contain cystine disulfides that are not critical to structure or activity. 

A reversible covalent modification that plants use extensively is the reduction of cystine disulfide bridges to sulf-hydryls. Many of the enzymes of photosynthetic carbohydrate synthesis are activated in this way (table 9.3). Some of the enzymes of carbohydrate breakdown are inactivated by the same mechanism. The reductant is a small protein called thioredoxin, which undergoes a complementary oxidation of cysteine residues to cystine (fig. 9.5). Thioredoxin itself is reduced by electron-transfer reactions driven by sunlight, which serves as a signal to switch carbohydrate metabolism from carbohydrate breakdown to synthesis. In one of the regulated enzymes, phosphoribulokinase, one of the freed cysteines probably forms part of the catalytic active site. In nicotinamide-adenine dinucleotide phosphate (NADP)-malate dehydrogenase and fructose-1,6-bis-... 

One erf the most convenient ways erf generating sulfhydryl groups is by reduction of indigenous disulfides. Many proteins contain cystine disulfides that are not critical to structure or activity. In some cases, mild reducing conditions can free one or more —SH groups for conjugation or modification purposes. The creation erf free sulf-hydryls in this manner allows for site-directed modification at a limited number of locations within the protein molecule. 

Ribulose phosphate kinase is active only when a cystine disulfide on the enzyme is reduced to two cysteines. An electron carrier, thioredoxin, carries out this reduction, and is then itself reduced by electrons from NADPH. Because the action of Photosystems I and II forms NADPH, this reduction ensures that ribulose bisphosphate is made only when enough light exists to support Photosynthesis. In other words, the light and dark reactions are coupled. 

The mechanism of disulfide reduction by phosphines is hypothesized to involve a stable intermediate containing a sulphur-phosphorous bond (6). Beta elimination would yield the phosphine sulfide and dehydroalanine. The formation of relatively stable adducts between cystine-containing peptides and the reagent was confirmed by mass spectrometry for several peptides with the major adduct representing one reagent molecule per cystine residue. 

Independently whether partial oxidation of Met residues occurs during the synthesis or whether Met(O) derivatives are purposely used, the sulfoxide has to be quantitatively reduced in the final steps to regenerate the methionine peptides. For this purpose, various reagents were proposed for the reduction in aqueous solution upon final deprotection and in organic solvents generally prior to the deprotection step in the presence or absence of cystine disulfides. 

Ruegg, U. T. and Rudinger, J., 1977. Reductive cleavage of cystine disulfides with tributylphosphine. Methods in Enzymology 47, 111-116. Shepherd, J. A., Waigh, R. D. and Gilbert, P., 1988. Antibacterial action of 2-bromo-2-nitropropane-l,3-diol (Bronopol). Antimicrob. Agents... 

Disulfides. As shown in Figure 4, the and h-chains of insulin are connected by two disulfide bridges and there is an intrachain cycHc disulfide link on the -chain (see Insulin and other antidiabetic drugs). Vasopressin [9034-50-8] and oxytocin [50-56-6] also contain disulfide links (48). Oxidation of thiols to disulfides and reduction of the latter back to thiols are quite common and important in biological systems, eg, cysteine to cystine or reduced Hpoic acid to oxidized Hpoic acid. Many enzymes depend on free SH groups for activation—deactivation reactions. The oxidation—reduction of glutathione (Glu-Cys-Gly) depends on the sulfhydryl group from cysteine. 

Dithiothreitol (DTT) and dithioerythritol (DTE) are the trans and cis isomers of the compound 2,3-dihydroxy-1,4-dithiolbutane. The reducing potential of these versatile reagents was first described by Cleland in 1964. Due to their low redox potential (—0.33 V) they are able to reduce virtually all accessible biological disulfides and maintain free thiols in solution despite the presence of oxygen. The compounds are fully water-soluble with very little of the offensive odor of the 2-mercaptoethanol they were meant to replace. Since Cleland s original report, literally thousands of references have cited the use of mainly DTT for the reduction of cystine and other forms of disulfides. 

Small molecules containing disulfide bonds (such as cystine-containing peptides) may be reduced and isolated simply by removing the immobilized reductant. Separation of reduced molecules from reductant is much more difficult if a soluble reducing agent is used with low-molecular-weight disulfides. 

The A and B peptide chains in insulin are linked through disulfide bridges. Their presence was suspected from the change in molecular weight which followed the reduction of insulin. For quantitative analyses the S-S bridges had to be broken. Sanger, following the approach used by Toennies and Homiller (1942), oxidized the protein with performic acid, so that the half-cystines were converted to cysteic acid. After oxidation, insulin could be separated into its A and B chains, the A peptide with 20 amino acid residues and the B with 30. 

It appeared probable that the reduction of disulfides to mercaptans by means of yeast would be particularly easy, since the two substances can be interconverted quite smoothly by ordinary chemical means. This prediction did not prove to be quite correct. True, Neuberg and Schwenk could reduce a disulfide by means of yeast, but they could not do so with the expected ease. Ethyl disulfide was chosen for the experiment because its boiling point (151°) differs very much from that of ethyl mercaptan (36°). Yeast which has been killed by boiling does not convert the disulfide into the mercaptan, but fermenting yeast does in view of the physiological importance of the disulfide group in cystine and glutathione, this observation is worthy of note. 

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