Disulfide bonds in proteins

The thioredoxin domain (see Figure 2.7) has a central (3 sheet surrounded by a helices. The active part of the molecule is a Pa(3 unit comprising p strands 2 and 3 joined by a helix 2. The redox-active disulfide bridge is at the amino end of this a helix and is formed by a Cys-X-X-Cys motif where X is any residue in DsbA, in thioredoxin, and in other members of this family of redox-active proteins. The a-helical domain of DsbA is positioned so that this disulfide bridge is at the center of a relatively extensive hydrophobic protein surface. Since disulfide bonds in proteins are usually buried in a hydrophobic environment, this hydrophobic surface in DsbA could provide an interaction area for exposed hydrophobic patches on partially folded protein substrates. 

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,). 

Many extracellular proteins like immunoglobulins, protein hormones, serum albumin, pepsin, trypsin, ribonuclease, and others contain one or more indigenous disulfide bonds. For functional and structural studies of proteins, it is often necessary to cleave these disulfide bridges. Disulfide bonds in proteins are commonly reduced with small, soluble mercaptans, such as DTT, TCEP, 2-mercaptoethanol, thioglycolic acid, cysteine, etc. High concentrations of mercaptans (molar excess of 20- to 1,000-fold) are usually required to drive the reduction to completion. 

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. 

Konigsberg, W. (1972) Reduction of disulfide bonds in proteins with dithiothreitol. In Methods in Enzymology, (C.H.W. Hirs, and S.N. Timaseff, eds.), Vol. 25 p. 185. Academic Press, New York. 

Traut, R.R., Casiano, C., and Zecherle, N. (1989) Cross-linking of protein subunits and ligands by the introduction of disulfide bonds. In Protein Function—A Practical Approach (T.E. Creighton, ed.), pp. 101-133. IRL Press at Oxford University, Oxford. 

Schnaible, V. Wefing, S. Resemann, A. Suckau, D. Biicker, A. Wolf-Kiimmeth, S. Hoffmann, D. Screening for Disulfide Bonds in Proteins by MALDI in-Source Decay and LlFT-TOF/TOF-MS. Anal Chem. 2002, 74, 4980-4988. 

Thompson, E. O. P., and I. J. O Donnell Quantitative reduction of disulfide bonds in proteins using high concentrations of mercapto-ethanol. Biochim. Biophys. Acta 53, 447 —449 (1961). 

The same authors [98] have presented a method for the estimation of the number of electroactive disulfide bonds in proteins adsorbed on mercury. The developed approach was based on the assumption that electroactive disulfides are located in more hydrophobic regions of the protein molecule. 

FIGURE 3-26 Breaking disulfide bonds in proteins. Two common methods are illustrated. Oxidation of a cystine residue with performic acid produces two cysteic acid residues. Reduction by dithiothreitol to form Cys residues must be followed by further modification of the reactive —SH groups to prevent re-formation of the disulfide bond. Acetylation by iodoacetate serves this purpose. 

In neural cells, the redox status is controlled by the thioredoxin (Trx) and glutathione (GSH) systems that scavenge harmful intracellular ROS. Thioredoxins are antioxidants that serve as a general protein disulphide oxidoreductase (Saitoh et al., 1998). They interact with a broad range of proteins by a redox mechanism based on the reversible oxidation of 2 cysteine thiol groups to a disulphide, accompanied by the transfer of 2 electrons and 2 protons. These proteins maintain their reduced state through the thioredoxin system, which consists of NADPH, thioredoxin reductase (TR), and thioredoxin (Trx) (Williams, Jr. et al., 2000 Saitoh et al., 1998). The thioredoxin system is a system inducible by oxidative stress that reduces the disulfide bond in proteins (Fig. 7.4). It is a major cellular redox system that maintains cysteine residues in the reduced state in numerous proteins. 

Reactions of sulfite and bisulfite with biochemical compounds are shown in Table II. Sulfite has been used frequently as a reagent for cleaving disulfide bonds in proteins (6, 9). Such a reaction may participate in the scheme of sulfur dioxide toxicity. 

The formation of disulfide bonds in proteins synthesized in vitro can be followed by measuring enzymatic activity or by an increased mobility compared to the reduced protein during SDS-PAGF. This increased mobility arises from the fact that, as disulfide-bonded proteins are intra-molecularly cross-linked, they form a more compact structure and occupy a smaller hydrodynamic volume compared to the reduced protein (Gold-enberg and Creighton, 1984). An illustration of this increase in mobility is shown in Fig. 2. Here the mRNA for preprolactin was translated in a cell-free system optimized for the formation of disulfide bonds, and then analyzed by SDS-PAGF. The translocated protein forms disulHde bonds under these conditions whereas the protein synthesized under the same conditions but in the absence of microsomal membranes does not form disulfide bonds. Thus the nascent protein must be translocated into microsomal vesicles for disulfide bond formation to occur. 

Yen TY, Joshi RK, Yan H, Seto NOL, Palcic MM, Macher BA. Characterization of cysteine residues and disulfide bonds in proteins by liquid chromatography/electrospray ionization tandem mass spectrometry. J. Mass Spectrom. 2000 35 990-1002. 

Reagents for Rapid Reduction of Disulfide Bonds in Proteins... 

Table I. Comparisons of Rate Constants for Reduction of Disulfide Bonds in Proteins Using Dithiol Reagents (DTT, BMS, DMH) ...

Table I shows a comparison of the apparent rate constants for the reduction of disulfide bonds in proteins using BMS, DMH and DTT. BMS and DMH reduce the disulfide bonds in proteins at pH 7 significantly faster than does DTT.

In the domain of food industries, EED was used to reduce oxygen in fruit juice [46], to extract cytoplasmic proteins from alfalfa [47,48], to coagulate proteins [49], and to reduce disulfide bonds in proteins [50]. These applications are based on the electrode redox reactions coupled with monopolar membrane action. 

Gekko et al. [59] have observed from the temperature dependenee of the molar volume that the reduction of disulfide bonds in proteins invariably leads to an increase of the thermal expansion. This is interpreted as a decrease in the internal cavity and an increase in the surface hydration. 

A second type of enzyme system which reduces disulfide bonds in proteins has been described. Protein-disulfide reductase (NAD (P)H) [NAD(P)H protein-disulfide oxidoreductase EC 1.6.4.4], purified from pea seeds (51), catalyzes the Reaction 2, shown on p. 111. 

Although there are no clear demonstrations of other enzyme groups (e.g., lyases, isomerases, ligases) affecting the functionality of proteins in foods, it is conceivable that they do. For example, protein disulfide isomerases could rearrange disulfide bonds in proteins to change their physical properties. 

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