Oxidation, disulfide bridge formation

Five oxidation procedures were compared for the disulfide bridge formation. 

CH2SH + 1/2 O2 -CH2-S-S-CH2 + H2O This reaction requires an oxidative environment, and such disulfide bridges are usually not found in intracellular proteins, which spend their lifetime in an essentially reductive environment. Disulfide bridges do, however, occur quite frequently among extracellular proteins that are secreted from cells, and in eucaryotes, formation of these bridges occurs within the lumen of the endoplasmic reticulum, the first compartment of the secretory pathway. 

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. 

The ability of coordinated NO to react with thiols has led to the suggestion of an alternative mechanism for activating guanylate cyclase. This involves nitroprusside oxidation of protein sulfhydryls to cross-link the protein with a disulfide bridge. For example, papain, which has an essential cysteine (cys-25) and glyceradehyde-3-phosphate dehydrogenase (cys-149) are both inhibited by nitroprusside with formation of [Fe(CN)5(NO)] and [Fe(CN)4NO] [132]. The suggested anaerobic reaction is ... 

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]. 

For the synthesis of double-stranded symmetrical and unsymmetrical monocystine peptides the formation of an intermolecular disulfide bridge is required. For homodimerization of cysteine peptides all the methods discussed in Section 6.1.1 can be applied taking into account the reactivity of the different oxidative agents toward sensitive amino acid residues present in the peptide sequences. Synthetic approaches based on the direct use of suitable cystine derivatives can be envisaged, at least for small-size peptides since disproportionation would in all cases retain the homodimeric structure 241... 

For the synthesis of heterodimeric cystine peptides where two different peptide chains are cross-linked by a disulfide bridge random co-oxidation of the two chains besides producing the heterodimer leads in the optimal case to the additional two homodimers in statistical distribution. Therefore, chemical control of the disulfide bridging via site-directed disulfide formation techniques is required since a thermodynamic control for generation of heterodimers is extremely difficult to achieve (see Section 6.1.5). 

A further development of the DMSO/H+ method for oxidation of cysteine peptides led to the cysteine-sulfoxide acid-catalyzed intermolecular disulfide formation with a second S-unprotected or acid-labile protected cysteine component as shown in Scheme 19. 1471 The protonation of the sulfoxide by TfOH in the case of 5(0)-Mob or TFA in the case of 5(0)-Acm derivatives provides electrophilicity to the sulfur atom to allow attack by the second S-unprotected cysteine component (formed by the fast deprotection of the 5-Mob group with TfOH in presence of dimethylsulfide) to generate in a site-directed manner the interchain disulfide bond. Although extensive experience with this method has not been accumulated for interchain disulfide bridging, it has been successfully applied for intrachain site-directed disulfide bond formation in chicken calcitonin-gene-related peptide.1 79 ... 

Many scorpion toxins, insect defensins, and enzyme inhibitors are cystine-rich polypeptides containing three to four disulfide bonds. In a large number of these toxins, two cystines are involved in the consensus Cys-(Xaa)1-Cys/Cys-(Xaa)3-Cys framework which is responsible for the common characteristic fold consisting of an a-helix and a two- or three-stranded antiparallel (3-sheet (a 3 3-fold or 3a 3 3-fold). For a review see ref[69]. The overall compact globular structures of these cystine-rich peptides contain the cystine stabilized a-helix motif (Section 6.1.5.1.2) which is further stabilized by a third disulfide bond between the N-terminus and the (3-strand adjacent to the helix and in some cases by an additional fourth disulfide bridge. Due to the presence of the cystine stabilized a-helix motif, a preferred initial formation of this motif followed by its stabilization via the additional disulfides was expected. However, in contrast to what was observed for the cystine peptides containing only the cystine stabilized a-helix motif, simple air oxidation is not successful. 

Oxidation of mono-cysteine peptides to the dimer is a straightforward reaction that can produce only the desired product. In the case of bis-cysteine peptides statistically the oxidation leads to the homodimers in parallel and antiparallel orientation as well as to the disulfide-bridged monomer and oligomers. When the two cysteine residues are placed in the adjacent position formation of homodimers is highly favored over the cyclic monomer (Section 6.1.5.1) and the product distribution depends strongly on the peptide concentration. Such a type of intermolecular disulfide bridging is present in bovine seminal ribonuclease, where an antiparallel alignment occurs at the interface of the dimer. 97 ... 

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