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Iodoacetate modification

Aqueous solutions of aequorin also emit light upon the addition of various thiol-modification reagents, such as p-quinone, Br2, I2, N-bromosuccinimide, N-ethylmaleimide, iodoacetic acid, and p-hydroxymercuribenzoate (Shimomura et al., 1974b). The luminescence is weak and long-lasting ( 1 hour). The quantum yield varies with the conditions, but seldom exceeds 0.02 at 23-25°C. The luminescence is presumably due to destabilization of the functional moiety caused by the modification of thiol and other groups on the aequorin molecule. [Pg.110]

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. [Pg.99]

The sulfur atom of methionine residues may be modified by formation of sulfonium salts or by oxidation to sulfoxides or the sulfone. The cyanosulfonium salt is not particularly useful for chemical modification studies because of the tendency for cyclization and chain cleavage (129). This fact, of course, makes it very useful in sequence work. Normally, the methionine residues of RNase can only be modified after denaturation of the protein, i.e., in acid pH, urea, detergents, etc. On treatment with iodoacetate or hydrogen peroxide, derivatives with more than one sulfonium or sulfoxide group did not form active enzymes on removal of the denaturing agent (130) [see, however, Jori et al. (131)]. There was an indication of some active monosubstituted derivatives (130, 132). [Pg.682]

When RNase-S was treated with iodoacetate at pH 6, both inactivation and histidine modification occurred 164). The modified histidine was in S-protein and was assumed to be His 119 since the sole product on analysis was 1-CM-His. In the absence of S-peptide only methionine modification occurred in S-protein. The loss of potential activity probably resulted from the reaction of the second of the two modifiable Met residues. The location of these residues in the sequence was not established. [Pg.688]

Bromoethylamine may undergo two reaction pathways in its modification of sulfhydryl groups in proteins (Fig. 84). In the first scheme, the thiolate anion of cysteine attacks the No. 2 carbon of 2-bromoethylamine to release the halogen and form a thioether bond (Lindley, 1956). This straightforward reaction mechanism is similar to the modification of sulfhydryls with iodoacetate (Section 4.2). In a two-step, secondary... [Pg.126]

Thus, iodoacetate has the highest reactivity toward sulfhydryl cysteine residues and may be directed specifically for—SH modification. If iodoacetate is present in limiting... [Pg.167]

The presence of imidazole groups in the active site region of human carbonic anhydrase B has, in fact, been demonstrated by chemical modification. Thus, bromoacetate reacts specifically with the 3 -N of a histidine residue to give a partially active monocarboxymethyl enzyme (65). The reaction depends on the initial combination of the bromoacetate ion with the anion binding site (65,83). In a detailed study, Bradbury (83) has shown that the irreversible reaction at saturation with iodoacetate... [Pg.177]

Figure 6. Modifications of imidazole groups (a) ethoxyformic anhydride (diethylpyrocarbonate) (b) iodoacetic acid (c) iodination. Figure 6. Modifications of imidazole groups (a) ethoxyformic anhydride (diethylpyrocarbonate) (b) iodoacetic acid (c) iodination.
Among the more important factors affecting reactions with proteins, pH is the most important since it controls the distribution of potentially reactive side chains between reactive and unreactive ionization states (see Table II). Iodoacetic acid is a commonly used reagent in protein modifications and serves as an example. At low pH values (such as 2-5)... [Pg.28]

The thiols in native lipoamide dehydrogenase are remarkably unreac-tive with other reagents only one thiol is at all reactive with DTNB or iodoacetate (61). Formation of the TNB-enzyme mixed disulfide is greatly increased by low concentrations (0.7 M) of guanidine hydrochloride (166). Its modification is associated with the destabilization of the enzyme in 1 Af guanidine hydrochloride which results in the slow reaction of 6 additional thiols. If the denaturant and excess DTNB are removed when the single thiol has reacted, the spectrum of enzyme-bound FAD is unmodified and the enzyme retains almost full activity. It is concluded that the thiol and the FAD are remote from one another in the protein (166). [Pg.123]

The characterization of proteins and their derivatives is outside the scope of this monograph. It is necessary to emphasize, however, that modification of native proteins frequently gives rise to complex mixtures of products. The complexity of the situation is not inunediately apparent solely from the stoichiometry of the modification reaction. The now classical case of the reaction of native bovine pancreatic ribonuclease A with iodoacetic acid at pH 5.5 may be cited in this context. One carboxymethyl group is introduced per molecule of... [Pg.11]

Reagents capable of modifying the histidyl residue with complete specificity are not available to date. Reaction with a-haloacids and amides at near neutral pH offers the best approach to the modification of histidine in native proteins. In a protein such as insulin, which contains neither methionyl nor cysteinyl residues, reaction with iodoacetate at pH 5.6 leads to the formation of a derivative in which the sole modification is the N-carboxymethylation of two histidyl residues (Covelli et al. 1973). [Pg.89]

Specific modification of Cys-46. Li and Vallee 86,87) and Harris 86) found that one cysteine residue per subunit may be selectively carboxymethylated with iodoacetate. The modified enzyme is inactivated and this cysteine residue, Cys-46 92), was suggested to be at the active site of the enzyme. The same residue in the S subunit is also especially reactive 20,94). The modification is preceded by anion binding of the iodoacetate and stimulated by the presence of imidazole 140,142,142). By using these facts and working with the crystalline enzyme, it is possible to achieve a highly specific and complete modification (ISO). X-ray studies of the carboxymethylated enzyme and the reaction mechanism of this modification are described in Section II,H. The carboxymethylation has been used to establish that both the EE 19) and SS 20) isozymes are active in u oxidations of fatty acids. [Pg.142]

This general anion binding site is especially interesting because of its involvement in the chemical modification of Cys-46 (Section II,E,l,a). The carboxymethylation of this residue is preceded by a reversible binding of iodoacetate to the enzyme (142,143). It has furthermore been shown that coenzyme, ADP-ribose, ADP, AMP, Pt(CN)4 and chloride ions but not 1,10-phenanthroline protects competitively against this reversible binding (130,140,14, 143). An inspection of the LADH model... [Pg.154]

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... [Pg.176]

Leskovac (109) has reported that a histidyl residue is also modified in m-MDH by iodoacetate. However, the labeled peptide isolated from this modification is completely distinct in composition from that found by Foster and Harrison (107) and by Sutton et al. (39) and does not appear to be related to any of the histidyl residues in the sequence of this enzyme (39). In the absence of further sequence data this discrepancy cannot be resolved at present. [Pg.393]


See other pages where Iodoacetate modification is mentioned: [Pg.109]    [Pg.111]    [Pg.120]    [Pg.183]    [Pg.183]    [Pg.872]    [Pg.101]    [Pg.47]    [Pg.118]    [Pg.119]    [Pg.167]    [Pg.562]    [Pg.15]    [Pg.11]    [Pg.43]    [Pg.28]    [Pg.292]    [Pg.204]    [Pg.313]    [Pg.214]    [Pg.28]    [Pg.90]    [Pg.118]    [Pg.147]    [Pg.25]    [Pg.1301]    [Pg.16]    [Pg.392]   


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Iodoacetalization

Iodoacetate

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