Disulfide bonds oxidation

Cleavage of all disulfide bonds. Oxidation with performic acid is commonly used. 

Lastly, if the number of cysteinyl residues in a protein is known from the amino acid sequence, one can infer how many disulfide bonds, or otherwise modified cysteinyl residues are present. If Ellman s assay cannot account for all of the cysteine residues predicted from the sequence, additional data, like high-accuracy MS, may be necessary to determine if the unreacted cysteinyl residues are involved in disulfide bonds, oxidized, or have undergone other chemical modifications. 

CH2SH + 1/2 O2 -CH2-S-S-CH2 + H2O Disulfide bonds form between the side chains of two cysfeine residues. Two SH groups from cysteine residues, which may be in different parts of the amino acid sequence but adjacent in the three-dimensional structure, are oxidized to form one S-S (disulfide) group. 

Disulfide bonds in proteins are generally stable and nonreactive, acting like bolts in the structure. However, oxidized DsbA is less stable than the reduced form and its disulfide bond is very reactive. DsbA is thus a strong... 

FIGURE 5.18 Methods for cleavage of disulfide bonds in proteins, (a) Oxidative cleavage by reaction with performic acid, (b) Reductive cleavage with snlfliydryl compounds. Disulfide bridges can be broken by reduction of the S—S link with snlfliydryl agents such as 2-mercaptoethanol or dithiothreitol. Because reaction between the newly reduced —SH groups to re-establish disulfide bonds is a likelihood, S—S reduction must be followed by —SH modification (1) alkylation with iodoac-etate (ICH,COOH) or (2) modification with 3-bromopropylamine (Br— (CH,)3—NH,). 

Figure 4-3. Oxidative cleavage of adjacent polypeptide chains linked by disulfide bonds (shaded) by per-formic acid (left) or reductive cleavage by 3-mercap-toethanol (right) forms two peptides that contain cysteic acid residues or cysteinyl residues, respectively.

Some proteins contain covalent disulfide (S— S) bonds that link the sulfhydryl groups of cysteinyl residues. Formation of disulfide bonds involves oxidation of the cysteinyl sulfhydryl groups and requires oxygen. Intrapolypeptide disulfide bonds further enhance the stability of the folded conformation of a peptide, while interpolypeptide disulfide bonds stabilize the quaternary structure of certain oligomeric proteins. 

Disulfide bonds between and within polypeptides stabilize tertiary and quaternary structure. However, disulfide bond formation is nonspecific. Under oxidizing conditions, a given cysteine can form a disulfide bond with the —SH of any accessible cysteinyl residue. By catalyzing disulfide exchange, the rupture of an S— bond and its reformation with a different partner cysteine, protein disulfide isomerase facilitates the formation of disulfide bonds that stabilize their native conformation. 

Insulin, a small protein of molecular mass 6000 daltons, is composed of two chains designated A and B. There are no reduced cysteine residues in insulin, but it contains three essential disulfide bonds two that crosslink the A and B chains, and one internal to the A chain to stabilize the overall tertiary stmcture. These disulfide bonds are cleaved in the presence of excess AuX4, leaving A and B chains that have cysteine residues that have become oxidized to sulfonic adds [119]. With smaller amounts of AuX4, a single disulfide bond will be attacked to form sulfinic acid [119]. The reaction is second order for AuCU while AuBr4 reacts too quickly for accurate monitoring. 

Witkiewicz, P.L. and Shaw, C.F. Ill (1981) Oxidative cleavage of peptide and protein disulfide bonds by gold(III) amechanism for gold toxicity. Journal of the Chemical Society, Chemical Communications, (21), 1111-1114. 

Disulfide bonds are hardly formed in cytosol of unmodified E. coli strains. Such enzymes can be expressed in E. coli Origami strain, in cells coexpressing helper proteins, such as PDI or DsbC, or they can be directed to the more oxidative periplasm. 

Cleland (1964) showed that DTT and DTE are superior reagents in reducing disulfide bonds in proteins (see previous discussion, this section). DTT and DTE have low oxidation-reduction potential and are capable of reducing protein disulfides at concentrations far below that required with 2-mercaptoethanol. However, even these reagents have to be used in approximately 20-fold molar excess in order to get close to 100 percent reduction of a protein. 

---