Reversible disulfide bond formation

Among the sundry approaches to tackle this issue [108], reversible disulfide bond formation was proven efficient in a seminal publication in 2001 [109]. Building on earlier work by Wulff and Schulze on chirally selective polystyrenes cross-... 

In 2000, the first example of ELP diblock copolymers for reversible stimulus-responsive self-assembly of nanoparticles was reported and their potential use in controlled delivery and release was suggested [87]. Later, these type of diblock copolypeptides were also covalently crossUnked through disulfide bond formation after self-assembly into micellar nanoparticles. In addition, the encapsulation of l-anilinonaphthalene-8-sulfonic acid, a hydrophobic fluorescent dye that fluoresces in hydrophobic enviromnent, was used to investigate the capacity of the micelle for hydrophobic drugs [88]. Fujita et al. replaced the hydrophilic ELP block by a polyaspartic acid chain (D ). They created a set of block copolymers with varying... 

Figure 2.41 Denaturing and self-reassembly of ribonuclease. The correct folding occurs only when disulfide bond formation is made reversible by the addition of P-mercaptoethanol (RSH) allowing the system to equilibrate (Reproduced from Section Key Reference by permission of The Royal Society of Chemistry).

Proteins, due to the complexity of their chemical structures, undergo oxidative modifications in subsequent stages which depend both on the presence of oxidation-susceptible groups and on steric availability of these groups for oxidant attacks (S25). Some oxidative structural modifications produced in proteins are common in various oxidants. Some modifications, such as chlorinated and nitrated protein derivatives produced in reactions with hypochlorite, peroxynitrite, and nitric dioxide, are specific for the oxidants employed. Certain oxidative protein modifications, such as interchain or intrachain disulfide bond formation or thiolation, are reversible and may be reduced back to the protein native form when oxidative stress is over (Dl). Other changes, such as sulfone formation, chlorination, and nitration, are irreversible and effect protein denaturation and promote its subsequent degradation. 

Furthermore, it should be mentioned that some of the amino acid side-chain modifications are actually reversible, including thiolation and disulfide bond formation, whereas others are irreversible, like methionine sulfone formation or nitration. 

Reverse phase HPLC (RP-HPLC) is the final cleanup step and leaves the protein in a volatile solvent suitable for lyophiHzation. Chemokines are functional when refolded and refolding involves cysteine oxidation and correct pairing in disulfide bonds. First, RP-HPLC is used to check for cysteine oxidation and disulfide bond formation. Then RP-HPLC is used to cleanup... 

The conformational change of the carrier in relation to the state of the loops facing the matrix was examined in more detail by studies on the effect on cysteine residues of copper-o-phenathroline(Cu(OP)2), which catalyzes disulfide bond formation between SH-groups [11]. Incubation of the particles with Cu(OP)2 specifically induced formation of a 60 kDa protein band detected by SDS-PAGE with decrease in the 30 kDa carrier band (Fig. 8), but no change in the protein band of the carrier in mitochondria (data not shown). The 60 kDa protein was reversed to the 30 kDa carrier by treatment with 2-mercaptoethanol (data not shown). Therefore, Cu(OP)2 specifically catalyzed the formation of an S-S bond between cysteine residues of the carrier acting from the matrix side. As the ADP transport... 

Scheme 18 The reversibility of the disulfide bonds allows for the formation of [2]- and [3]rotaxanes (33 and 34) under thermodynamic control... 

The above observations are consistent with a thermodynamically controlled process shown in Figure 9.17. Thus, when two strands, A and B, with complementary H bonding sequences and termini that can be reversible cross-linked, are present in the same solution under redox conditions, products A-A (or self-cyclized A ), B-B (or self-cyclized B ), and A-B, may be generated. Among these products, A-B gains the most stabilization from the newly generated, complementary intramolecular H bonds because of the formation of the two disulfide bonds. Thus,... 

Richardson-style diagram of the polypeptide backbone of the individual structure of AP-A [46] that is closest to the average over the whole molecule. The locations of the sulfurs in the three disulfide bonds (4-46, 6-36, and 29-47) are shown in CPK format. The locations of reverse turns found in more than half the NMR-derived structures (6-9, 25-28, and 30-33) are indicated by darker backbone shading. 

Catsimpoolas et al. (2) and Hermansson ( ) have found that the reaction leading to formation of the soluble aggregates is reversible. Recently, Takagi et al. ( ) found that hydrophobic interactions are primarily responsible for the formation of the soluble aggregates which are subsequently irreversibly insolu-bilized through intermolecular disulfide bond interchange. 

FIGURE 3-7 Reversible formation of a disulfide bond by the oxidation of two molecules of cysteine. Disulfide bonds between Cys residues stabilize the structures of many proteins. 

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