Protein disulfide bond reduction

Anfinsen, C. B., and E. Haber Studies on the reduction and re-formation of protein disulfide bonds. Journ. Biol. Chem. 236, 1361—1.363 (1961). 

Cline DJ, et al. New water-soluble phosphines as reductants of peptide and protein disulfide bonds reactivity and membrane permeability. Biochemistry 2004 43 15195. 

Disulfide (-S-S-) bonds play an important role in the structure and function of proteins. IgG molecules are comprised of two heavy and two light chains linked by interchain disulfide bonds. In addition, intrachain disulfide bonds are also present in IgGs. Alkyl thiols have been used to demonstrate the structural and functional role played by disulfide bonds. Reduction of IgGs with alkyl thiols under denaturing conditions results in separation of light and heavy chains. 

Fig. 17.7. Engineered protein designed for mechanochemistry studies, (a) A pair of cysteine residues introduced into the 127 protein (positions 32 and 75 suifur atoms as spheres) spontaneously form a buried disuifide bond. (b). In response to an unfolding force, the protein extends right up to the disuifide bond. Unfoiding exposes the disulfide bond to the solution, (c) Then, a nucieophiie such as DTT can initiate a Sn2 reaction, leading to the reduction of the disuifide bond and the concomitant extension of the amino acids that were trapped behind the disuifide bond. This sequence of events nnambiguousiy identifies individual disulfide bond reduction events, allowing for the stndy of a pulling force on a Sn2 chemicai reaction...

Chromophores can also be synthetically attached to specific protein groups, either to assist identification or incidental to blocking reactive fiinctionalities. One common chromophore is the pyridylethyl group added to cysteine using 4-vinylpyridine after disulfide bond reduction but prior to enzymatic digestion. The pyridylethyl cysteine has a strong absorbance maximum near 254 nm that permits easy on-line identification of Cys-containing peptides [16]. 

Disulfide bond reduction, which might also cause some release of structural constraints imposed by the disulfide bond and protein unfolding at the interface. Soy glycenin, which has limited film-forming properties due to its compact, stable, disulfide-linked structure [128] will be significantly more surface-active when some of its six disulfide-linked acidic and basic subunits have been reduced (Fig. 48). Reduction, oxidation-reduction, or oxidation of the amino acid cysteine to form an open thiol (sulfhydryl) groups. 

Disulfides. The introduction of disulfide bonds can have various effects on protein stability. In T4 lyso2yme, for example, the incorporation of some disulfides increases thermal stability others reduce stability (47—49). Stabili2ation is thought to result from reduction of the conformational entropy of the unfolded state, whereas in most cases the cause of destabili2ation is the introduction of dihedral angle stress. In natural proteins, placement of a disulfide bond at most positions within the polypeptide chain would result in unacceptable constraint of the a-carbon chain. 

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,). 

Protocol for the Complete Reduction of Disulfide Bonds within Protein Molecules... 

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. 

Ethylenimine may be used to introduce additional sites of tryptic cleavage for protein structural studies. In this case, complete sulfhydryl modification is usually desired. Proteins are treated with ethylenimine under denaturing conditions (6-8 M guanidine hydrochloride) in the presence of a disulfide reductant to reduce any disulfide bonds before modification. Ethylenimine may be added directly to the reducing solution in excess (similar to the procedure for Aminoethyl-8 described previously) to totally modify the —SH groups formed. 

Figure 6.2 The trifunctional reagent sulfo-SBED reacts with amine-containing bait proteins via its NHS ester side chain. Subsequent interaction with a protein sample and exposure to UV light can cause crosslink formation with a second interacting protein. The biotin portion provides purification or labeling capability using avidin or streptavidin reagents. The disulfide bond on the NHS ester arm provides cleavability using disulfide reductants, which effectively transfers the biotin label to an unknown interacting protein.

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