Alkylation of disulfide bonds

Since reductive S-alkylation of disulfide bonds introduces unnatural amino acid side chains into the protein and, therefore, cannot serve as a useful model for nutritional and toxicological studies, we initiated systematic studies of effects of thiols on the inhibitory process. Such thiols are expected to interact with inhibitor disulfide bonds via sulfhydryl-disulfide interchange and oxidation reactions (Friedman, 1973). 

Chemical modification by disulfide bond cleavage can markedly alter thermal stabilities or other properties of the inhibitors, since disulfide crosslinks help maintain the active conformations of many proteinase inhibitors (Laskowski and Sealock, 1971 Tschesche, 1974 Weder, 1981). Loss of thermal stability resulting from reductive alkylation of disulfide bonds has been observed for BPTI by H-NMR (WQthrich et al., 1980), for proteinase inhibitor I from potato by immunological assay (Plunkett and Ryan, 1980), and for STI, LBI, and chicken ovomucoid by DSC (Friedman et al., 1982c). 

The effects of a number of other perturbations on the interaction of RBP with TTR (and on the interactions of these proteins with their ligands retinol and thyroxine, respectively) have been explored. The other manipulations that have been examined include effects of changes in temperature and urea concentration and the effects of reduction and alkylation of disulfide bonds and of iodination (Raz et al., 1970) the effects of modifications of tyrosine, lysine, and tryptophan residues were also examined (Heller and Horwitz, 197S Horwitz and Heller, 1974b). Addition of 6 Af urea completely disrupted the RBP-TTR complex, markedly reduced the affinity of TTR for thyroxine, but did not interfere with the association of RBP with retinol. Iodination of isolated RBP decreased its affinity for TTR (Raz et al., 1970 Vahlquist, 1972 Vahlquist et al., 1973). However, it was found (Vahlquist, 1972 Vahlquist et al., 1973) that iodination of the RBP-TTR complex, followed by the dissociation of the complex and separate isolation of the two proteins, yielded iodinated RBP with full affinity for... 

These studies with retinoids have provided some information about the structural requirements of the retinol binding site on RBP. No information is, however, available about the amino acid residues in RBP that are involved in the binding site. Acetylation of lysine residues of RBP did not affect its binding of retinol (Heller and Horwitz, 1975). Modification of one of eight tyrosine residues and two of four tryptophan residues of RBP also had no effect on the retinol-RBP interaction (Heller and Horwitz, 1975 Horwitz and Heller, 1974b). The binding site was, however, disrupted by reduction and alkylation of disulfide bonds (Raz et al., 1970). 

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,). 

Cyanogen Iodide (ICN) has been used extensively for the cyanation of alkenes and aromatic compounds [12], iodination of aromatic compounds [13], formation of disulfide bonds in peptides [14], conversion of dithioacetals to cyanothioacetals [15], formation of trans-olefins from dialkylvinylboranes [16], lactonization of alkene esters [17], formation of guanidines [18], lactamization [19], formation of a-thioethter nitriles [20], iodocyanation of alkenes [21], conversion of alkynes to alkyl-iodo alkenes [22], cyanation/iodination of P-diketones [23], and formation of alkynyl iodides [24]. The products obtained from the reaction of ICN with MFA in refluxing chloroform were rrans-16-iodo-17-cyanomarcfortine A (14)... 

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. 

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]. 

To determine the number of disulfide bonds, the protein is reduced and alkylated as described in Section 8.5.1 by reacting it with either dithiothreitol or 2-mercaptoethanol, followed by reaction with iodoacetic acid. The molecular masses of the native protein (Mnat) and reduced and alkylated protein (Mr+a) are determined with MALDl-MS or ESl-MS. S-Carboxymethylation increases the mass of each cysteine residue by 59 Da. From the change in the molecular mass, the number of cysteine residues (Ncys) and hence the number of disulfide bonds (Ns-s) can be estimated using... 

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. 

Solution First, the number of free sulfhydryl groups must be calculated from the inaease in mass that resulted from alkylation of the protein. Therefore, A sh = (10,391 — 10,275)/58 = 2. The second step is to calculate the number of cysteine residues from the increase in mass upon reduction and alkylation reactions. Ncys = (10,865 — 10,275)/59 = 10. Finally, the number of disulfide bonds = (10 - 2)/2 = 4. 

Allow the reduced protein solution to cool to room temperature and add lAA from a 0.5 M stock solution to reach 20 mM concentration in the reduced cell lysate. Incubate the reaction for 30 min at room temperature in the dark to alkylate the cysteine residues. Modification of the protein thiol groups by alkylation prevents reformation of disulfide bonds due to oxidation upon exposure to air. 

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... 

As already mentioned, dehydroalanine is the postulated reactive precursor for lysinoalanine. Direct evidence for dehydroalanine reactivity was obtained by Friedman et al. (1977). They showed that dehydroalanine derivatives convert lysine side chains in casein, bovine serum albumin, lysozyme, wool, or polylysine to lysinoalanine residues at pH 9 to 10. Related studies showed that protein SH groups generated by reduction of disulfide bonds are completely alkylated at pH 7.6 to lanthionine side chains. These studies demonstrate that lysinoalanine and lanthionine residues can be introduced into a protein under relatively mild conditions, without strong alkaline treatment. They also imply that it should be possible to explore nutritional and toxicological consequences of lysinoalanine and lanthionine consumption in the absence of racemiza-tion (see below). 

The stannylenes from either source will insert into the Sn- Sn, Sn-R, or Sn-H bonds of organotin compounds, and react with alkyl halides, disulfides, or peroxides as shown in the reaction scheme below, but only the stannylenes that are generated photolytically will react with carbonyl compounds, and it appears that the stannylenes may exist in two forms, perhaps related as singlet and triplet, or a com-plexed and uncomplexed species. 

The extent of formation of protein disulfides with time was determined by withdrawing aliquots which were acidified to pH 5.5 and alkylated with N-ethylmaleimide. The disulfide content of the peptide was determined after its isolation. Formation of two intrapeptide disulfide bonds proceeded at the same rate (within experimental error) as formation of the first two disulfides in reduced lysozyme. The first-order rate constant for these two processes (0.5 min-1) was eight times that describing the rate of oxidation of reduced lysozyme in the presence of 6 M guanidinium chloride, suggesting substantial specificity in the process in absence of denaturant. An additional indication of specificity was the finding that 13-105 reached its maximum of two —S—S— bonds in less than 20 minutes, retaining one reduced thiol from 20 to 240 minutes. For subsequent studies this material was S-alkylated with N-ethylmaleimide. 

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