Disulfide bonds reduction/alkylation

Figure 4.26. Disulfide-Bond Reduction. Polypeptides linked by disulfide bonds can be separated by reduction with dithiothreitol followed by alkylation to prevent reformation.

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. 

Stable, monovalent subunits of SNA and MAL have been prepared by reduction of disulfide bonds and alkylation with 4-vinylpyridine. These derivatives have proved to be useful reagents for the study of cell surface glycoconjugates by flow cytometry [248,249]. 

In general, the digestion process has to be optimized to achieve maximum efficiency based on a number of parameters affecting the enzymatic reaction that include (i) solubilization and denaturation of proteins, (ii) reduction of disulfide bonds, (iii) alkylation of reduced cysteines, and (iv) digestion conditions. 

Reduction of Disulfide Bonds and Alkylation of Reduced Cysteines... 

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,). 

Complete reduction of all the disulfide bonds in peptides and proteins is generally achieved by treatment with excesses of dithiothreitol (DTT) under denaturing conditions. Due to the highly differentiated redox potentials of the disulfides, selective reduction of single disulfide bonds can also be achieved. Subsequent alkylation of the thiol groups generated by this partial reduction leads to stable derivatives, which are useful in the analysis of disulfide connectivities. 

Antigen unmasking on sections of paraffin-embedded tissues can be accomplished by reduction of disulfide bonds by treatment with 2-mercaptoethanol, followed by alkylation with sodium iodoacetate to prevent the bonds from reforming. This method has been used for unmasking a Kunitz protease inhibitory domain epitope of Alzheimer s amyloid precursor protein in human brain (Campbell et al., 1999). Sections are reduced with a mixture of 0.14 M 2-mercaptoethanol in 0.5 M Tris-HCl (pH 8.0) and 1 mM EDTA for 3 hr in the dark at room temperature. After being washed for 3 min in distilled water, the sections are treated with a mixture of 250 mg/ml iodoacetic acid in 0.1 M NaOH, diluted 1 10 in 0.5 M Tris-HCl (pH 8.0) and 1 mM EDTA for 20 min in the dark. 

Reduction of disulfide bonds followed by alkylation of the free cysteines to prevent re-oxidation, while not essential for the digestion of most proteins, generally gives better results. This is due to increased susceptibility of the reduced/ alkylated protein to tryptic digestion and the absence of any disulphide-linked peptides (which are not matched in the database search) from the PMF data (6). 

Disulfide linkages may be broken either oxidatively or reductively. The former method involves the treatment of the protein with performic add, which converts all disulfide bonds into cysteic add residues. This procedure is usually performed before a protein is hydrolyzed for amino add analysis. Cystine and cysteine are then determined as cysteic add. The reductive cleavage of disulfide bonds involves the treatment of the protein with mercaptoethanol (SH-CH2-CH2-OH), followed by the alkylation of the newly formed -SH groups. The complete disruption of all secondary interactions (that is, complete denaturation) can be achieved in most proteins with 6 M guanidine hydrochloride and 0.1 M mercaptoethanol or 8 M urea and 0.1 M mercaptoethanol. 

Cleavage of disulfide bonds occurs before hydrolysis of the protein into peptides. Disulfide bonds may be cleaved oxidatively, or they may be reduced and alkylated. Treatment of the native protein with performic acid, a powerful oxidizing agent, breaks disulfide bonds and converts cystine residues to cysteic acid (Figure 3-11). Reduction of the disulfide linkage by thiols, such as d-mercaptoethanol, yields reactive sulfhydryl groups. These groups may be stabilized by alkylation with iodoacetate or ethyleneimine to yield the carboxymethyl or aminoethyl derivative, respectively. 

Reduction-alkylation of proteins. The molecular mass measurement of the target protein before and after this step provides information on the number of cysteine residues and disulfide bonds. 

Because cysteine residues are prone to autooxidation, proteins that contain disulfide bonds or free sulfhydryl groups must be subjected to reduction-alkylation process prior to cleavage. Alkylation converts sulfhydryl groups to stable derivatives. Also, during the reduction-alkylation procedure, the three-dimensional structure of proteins is disrupted to allow more cleavage sites to be accessible. Proteins are first reduced by treatment with 2-mercaptoethanol or dithiothreitol to convert disulfide bonds to free sulfhydryl group ... 

Example 9.1 The molecular mass of a protein is 10,275 Da. Upon reduction with dithiothreitol and alkylation with iodoacetic acid, its mass increased to 10,865 Da. In a separate experiment, the protein was treated only with iodoacetic acid. The molecular mass of the protein was found to be 10,391 Da. Calculate the number of disulfide bonds in this protein. 

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