Alkylation with iodoacetate

In addition to the alkylation with iodoacetate (Eq. 3-24), sulfhydryl groups can react with N-ethylmale-imide (Eq. 3-39).281 This reaction blocks the SH groups irreversibly and has often been used in attempts to establish whether or not a thiol group plays a role in the functioning of a protein. Loss of function in the... 

The probable general structure of the dimers was established in elegant experiments by Fruchter and Crestfield 381) involving alkylation with iodoacetate. The two isomeric dimers referred to above behave identically in these reactions. The two active sites in the dimers behave just like that of the monomer. Histidines 12 and 119 both react, but the reactions are mutually exclusive. The proposed structure is outlined in Fig. 19. The tail of one monomer combines with the body of the other and vice versa. The His 12 and 119 pairs are now on separate molecules. When the dimers, fully inactivated by reaction with iodoacetate, are dissociated by heating at neutral pH, the following monomers would be expected native RNase (active), CM-His-12-RNase (inactive) CM-His-119-RNase (inactive), and di-CM-His-12-His-119-RNase (inactive). These were, in fact, found. About 2b% activity reappeared from the inactive dimer. Equally important the di-CM compound was found. This material... 

Figure 2. Tryptic map of human relaxin B-chain. The peptide was reduced with dithiotreitol and alkylated with iodoacetic acid before digestion with trypsin. The chromatography was performed on a Vydac Cis column using TFA-containing mobile phases, and eluted with an acetonitrile linear gradient.

Lovenberg, Buchanan, and Rabinowitz found that treatment of ferredoxin with iodoacetate or N-ethylmaleimide in either the presence or absence of 8 M urea had no effect on its spectral characteristics. Less than 1 mole of carboxymethyl cysteine was formed per mole of protein when native ferredoxin was treated with iodoacetate-1-C14 (Table 10). Sobel and Lovenberg (96) showed recently that C14-iodoacetate did not react appreciably with reduced ferredoxin. However, Table 10 shows that if ferredoxin was treated with 2-mercaptoethanol in 8 M urea, it was alkylated with iodoacetate. This demonstrated that the half-cystine residues of native ferredoxin were not present as free sulfhydryls, and the mercurial titration data given above showed that they were not present as disulfides. The two observations were consistent, therefore, with a structure in which the half-cystine residues are present as cysteine and are bonded with the iron by a sulfide bridge. 

In a study of the reducing action of various thiols at 0.1 M concentration in aqueous solutions of alcohols (Maclaren, 1962), benzyl mercaptan was found to be the most effective. Maximum reduction exceeding 90 % was obtained in 20 % propanol solution, and under these conditions the wool remained intact. If 5 M Nal was incorporated in the solution approximately 65 % of the protein was extracted in 48 hr at 20°C under essentially neutral conditions (Maclaren, unpublished observations, 1962). This protein could be alkylated with iodoacetate and fractioned into low-sulfur and high-sulfur fractions using the methods of Gillespie. 

Nitrogens 1 and 3 in the imidazole ring of histidyl residues in proteins may be alkylated with iodoacetic acid (generally in a much slower reaction than alkylation of cysteinyl residues) to give three carboxy-methyl derivatives 1-carboxymethylhistidine, 3-carboxymethyl-histidine and 1,3-dicarboxymethylhistidine ( 3.4). In general, the 3-carboxymethyl derivative is formed most rapidly. These derivatives are stable to acid hydrolysis under the usual conditions (but excess reagent must be removed) and may be analyzed on the long column of most analyzers as described below. 

Auto-oxidation of cysteine residues during cleavage of the disulfide bridge-containing proteins is a potential concern. This concern can be addressed by first reducing those proteins at alkaline pH ( 8.0) with either 2-mercaptoethanol or dithiothreitol (Equation (1)) and then alkylating with iodoacetic acid to S-carboxymethyl derivatives (Equation (2)) The reduction-alkylation process also disrupts the 3D structure of proteins to allow more sites accessible for cleavage. 

Diagnostic losses of certain neutral species are commonly observed from b- and j-ions. For example, Ser, Thr, Asp, and Glu side chains exhibit prominent loss of water (—18 Da), and the Asn-, Gin-, Lys-, and Arg-containing ions similarly show abundant loss of ammonia (—17 Da). The loss of 48 Da (HSCH3) is observed from Met-containing sequence ions, but if Met is in oxidized form, the mass of the expelled neutral ion becomes 64 Da. The Cys-containing ions show a loss of 34 Da (H2S), which shifts to 92 Da (HSCH2COOH) if the precursor protein is alkylated with iodoacetic acid prior to digestion. The peptides that contain a basic residue at the C-terminus are likely to expel the C-terminal residue to produce the (h , + OH) ion. 

The structures of ebumaminol (155) and larutensine (154) have been confirmed by a synthesis reported by Lounasmaa from the previously available indoloquinolizidine ester 154 (Scheme 8). Successive reduction, acetylation and Fuji oxidation of 156 yielded the enamine 157 which was alkylated with iodoacetic ester followed by NaBH4 reduction to give a mixture of four products. Treatment of two of these, the epimeric esters 158, with ethanolic sodium ethoxide resulted in cyclization to 18-hydroxyebumamonine (159) accompanied by its C(20) epimer. Reduction of 18-hydroxyebumamonine furnished ( )-ebumaminol and 16-epiebumami-nol (160) which on overnight treatment with acid gave ( )-larutensine [114]. 

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. 

Glyceraldehyde-3-phosphate dehydrogenase, an enzyme in the glycolytic pathway (Chapter 8), is inactivated by alkylation with iodoacetate. Enzymes that use sulfhydryl groups to form covalent bonds with metal cofactors are often irreversibly inhibited by heavy metals (e.g., mercury and lead). The anemia in lead poisoning is caused in part because of lead binding to a sulfhydryl group of fer-rochelatase. Ferrochelatase catalyzes the insertion of Fe2+ into heme. 

Two of the cysteine residues are especially reactive toward chemical modification. Thus, one residue per subunit is selectively alkylated with iodoacetate (55) and a different one with butylisocyanate 406,407). In both cases the enzyme is inactivated and protected by the coenzyme against modification, suggesting that these residues are at the active sites of the enzyme. The two residues are now known 12,137) to be homologous to the two reactive cysteine residues in the horse enzyme, Cys-46 and Cys-174 (Section II,E,l,a), which are ligands to the active site zinc atom (Section II,C,3,b). A number of other reagents, apart from reactive coenzyme analogs, have also been shown to modify essential cysteine residues, i.e., probably either of these residues. Thus, one cysteine residue... 

Fig. 7.8. The -SH groups are protected by alkylation with iodoacetic acid to prevent re-oxidation.

Example 8.1 A protein with two sulfide bonds and three cysteine residues is first reduced with 2-mercaptoethanol and then alkylated with iodoacetic acid. By how much will the mass of the protein increase ... 

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. 

Ribonuclease A contains several disulfide bonds but no free sulfhydryl group. The molecular mass of the protein measured before and after reduction and alkylation (with iodoacetic acid) was found to be 13,682 and 14,155 Da, respectively. How many disulfide bonds does this protein contain ... 

In the case of amino acid analysis, the quantification of cysteine can be difficult because it is oxidized to cystine during acid hydrolysis. To circumvent this problem, cysteine can be oxidized to cysteic acid with performic acid prior to analysis. Alternatively, cysteine can be converted to the pyridyl ethyl derivative and subsequently detected by postcolumn reaction with ninhydrin. Still another method involves the production of carboxymethyl cysteine following alkylation with iodoacetic acid. All of these cysteine derivatives can be separated by either reversed-phase precolumn or ion-exchange postcoT umn methods. 

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