Reduction of disulphide bonds

Bockle, B. and Muller, R. (1997). Reduction of disulphide bonds by Streptomyces pactum during growth on chicken feathers. Appl. Environ. Microbiol. 63,... 

Finally, disulphide bonds can be located by hydrolysing a protein to a mixture of peptides using either a proteinase or a specific chemical method of cleavage and the mixture can be analysed directly by fast-atom bombardment mass spectrometry (Chapter 3) and again after reduction of disulphide bonds (Yazdanparast et al., 1987). By identifying those peaks which disappear as a result of reduction and new peaks with appropriate masses that have taken their place, it is simple to assign disulphide bonds to the relevant amino-acid sequences. 

For preparing whole ricin IT, the intact toxin is normally derivatized with a bifunctional cross-linking agent before being mixed with antibody prepared for cross-linking by partial reduction of disulphide bonds [183]. 

The effects of dimethyl sulphoxide, lithium bromide, guanidinium chloride, sodium dodecyl sulphate, and urea on lysozyme have been studied using Raman spectroscopy. The spectrum observed was found to depend on the denaturant used, suggesting there is not a unique denatured state for lysozyme. An analysis of the interaction of sodium dodecyl sulphate with lysozyme has been published. A kinetic study of the denaturation and subsequent reduction of disulphide bonds in lysozyme has been made using rapid ultrasonic absorption measurements. 

Reduction of disulphide bonds followed by alkylation of the free cysteines to prevent reoxidation, while not essential for the digestion of most proteins, generally gives better results. This is due to increased susceptibility of the reduced/alkylated protein to tryptic digestion and the absence of any disulphide-linked peptides (which are not matched in the database search) from the PMF data (6). 

PH3 itself is cytotoxic and used as a fumigant against pests in stored products. It inhibits mitochondrial respiration but the details of the process are unclear. The oxidation pathways for PH3 differ significantly from those of tertiary phosphines PR3 and for these the available pathways depend on the substituents R. Pathways involving radicals usually lead to p-sdssion when R = aryl but a-scission when R = alkyl. The autoxidation of alkyl phosphines is usually rapid compared to aryl phosphines. Similar differences exist in the reduction of disulphide bonds the alkyl derivatives are more reactive. 

It is usual to effect cleavage of disulphide bonds by reduction or oxidation. Addition of a large excess of a thiol such as 2-mercaptoethanol or 1,4-dithiothreitol to a polypeptide reduces cystine residues to cysteine (Scheme 5.1). In order to prevent reoxidation in air, the generated thiol groups are blocked, usually by reaction with iodoacetic acid. The product yields S -carboxymethylcysteine (5.9) on hydrolysis for amino-acid analysis. Alternatively, oxidative cleavage of disulphide bonds can be achieved with performic acid each half of the cysteine residue is converted into a residue of cysteic acid (5.10). 

A development reported recently [519] involves reduction of the cystine disulphide bonds in wool with either thioglycolic acid or tetrakis(hydroxymethyl)phosphonium chloride to form thiol groups, followed by crosslinking with bifunctional reactive dyes. This gave improved insect resistance but had adverse effects on physical properties such as strength, shrinkage and stiffness, thus limiting the potential of the process for commercial use. 

The method described here is based on two reactions first, the reduction of the disulphide bond between Hey and other thiols or the cysteine residue of proteins by the reducing compound tri-n-butylphosphine (Fig. 2.2.2a) followed by the reaction of Hey and other thiols with the flourogenic thiol-specific reagent ammonium... 

Polarography has been successfully applied to the investigation of structural problems involving sulphur compounds. The presence of a disulphide bond has been established by means of the polarographic reduction waves of cytochrome C (156) and lipoic acid (757), and in cyclic disulphides of the oxytocine and vasopressine type (158). The elucidation of the process responsible for the reduction wave of lipoic acid was carried out by comparison with reduction waves of cyclic disulphides, where the disulphide bond was incorporated into rings of various size. The similarity indicated that in lipoic acid an S—S bond which is a part of a larger cyclic system is reduced. 

Another possibility to introduce two functional groups at a predetermined distance was investigated by polymerising the disulphide 6 under imprinting conditions (see entry k). Subsequent reduction of the disulphide bond with diborane provides polymers with two closely positioned mercapto groups (see Scheme 4.I1I). 

Aminopenicillanic acid (8) was converted to 6(5)-bromopenicillanic acid by trapping of the diazo-intermediate with hydrogen bromide. Esterification of the dicyclohexylamine salt (93) with p-methoxybenzyl bromide, followed by oxidation, afforded the sulphoxide (94) in 60% yield from 6-APA. Elaboration of this sulphoxide to the disulphide (96) was effected by the procedure established by Kamiya et al. [98] the sulphenic acid (95), formed by heating the sulphoxide to reflux in toluene, was intercepted by reaction with 2-mercaptobenzothiazole to yield the disulphide (96). The latter was transformed by base-catalysed double bond isomerization to the conjugated ester disulphide (97) [95% yield from (94)]. Reductive formylation of disulphide (97) then provided the formylthio-derivative (98). Cyclization of the oxalimide (99), obtained by ozonolysis of... 

Free formaldehyde is released from the above reaction, which in turn acts as a reducing agent on the polymer disulphide bonds to bring about a reduction into thiol with the formation of formic acid ... 

Out of the above bonds, the disulphide bond (covalent bond) is the strongest and cannot be affected by solvent, pH, temperature and salts whereas the above conditions. The disulphide bond can be split and reformed by oxidation/reduction respectively. The tertiary structure gains special importance in the case of enzymes. 

Other chemical degradation pathways include -elimination, which can lead to racemization and disulphide exchange reactions. The amino acids that may undergo P-elimination include Cys, Ser, Thr, Phe, and Lys (40) and occur especially at alkaline pH (64). The reduction and oxidation of the disulphide bonds are often accompanied by a considerable change in the protein conformation (23,33). An example is the secondary structure of insulin that is disrupted or completely lost when the disulphide bonds are broken (23). The disulphide bond disruption or interchange can also result in an altered three-dimensional structure and therefore a possible loss of activity (40) or aggregation (29). For further details, the reader is directed to the following reviews and book chapters (30,40,62,65). 

P. A. Price, W. H. Stein, and S. Moore. Effect of divalent cations on the reduction and reformation of the disulphide bonds of deoxyribonuclease. J. Biol. Chem. 244 929-932 (1969). 

A putative y-amide derivative was also prepared from MTX by reaction with cystamine in water solution in the presence of EDC hydrochloride, followed by reduction of the disulphide bond with excess 2-mercaptoethanol [328]. The resulting thiol (VIII.220) was then allowed to react with... 

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