Sulfhydryls with Iodoacetate

The relative reactivity of a-haloacetates toward protein functionalities is sulfhydryl imid azolyl thioether amine. Among halo derivatives the relative reactivity is I Br Cl F, with fluorine being almost unreactive. The a-haloacetamides have the same trend of relative 

Dissolve the sulfhydryl-containing protein or macromolecule to be modified at a concentration of l-10mg/ml in 50mM Tris, 0.15M NaCl, 5mM EDTA, pH 8.5. EDTA is present to prevent metal-catalyzed oxidation of sulfhydryl groups. The presence of Tris, an amine-containing buffer, should not affect the efficiency of sulfhydryl modification. Not only do amines generally react slower than sulfhydryls, the amine in Tris buffer is of particularly low reactivity. If Tris does pose a problem, however, use 0.1M sodium phosphate, 0.15M NaCl, 5mM EDTA, pH 8.0. 

Add iodoacetate to a concentration of 50mM in the reaction solution. Alternatively, add a quantity of iodoacetate representing a 10-fold molar excess relative to the number of —SH groups present. An estimation of the sulfhydryl content in the protein to be modified can be accomplished by performing an Ellman s assay (Chapter 1, Section 4.1). Readjust the pH if necessary. To aid in adding a small quantity of iodoacetic acid to the reaction, a concentrated stock solution may be made in the reaction buffer, the pH re-adjusted, and an aliquot added to the protein solution to give the desired concentration. 

Mix and react for 2 hours at room temperature. To avoid the possibility of methionine modification, limit the reaction to 30 minutes. 

Purify the modified protein from excess iodoacetate by dialysis or gel filtration. 

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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... 

Another approach uses reactive alkyl halogen compounds containing a terminal carboxylate group on the other end to form spacer arms off the dextran polymer from each available hydroxyl. In this manner, Brunswick et al. (1988) used chloroacetic acid to modify the hydroxyl groups to form the carboxymethyl derivative. The carboxylates then were aminated with ethylene diamine to create an amine-terminal derivative (Inman, 1985). Finally, the amines were modified with iodoacetate to form a sulfhydryl-reactive polymer (Figure 25.14). 

Figure 25.18 An amine-derivative of dextran may be coupled with iodoacetic acid using a carbodiimide reaction to produce a sulfhydryl-reactive iodoacetamide polymer.

Haloacetyl Method The haloacetyl method uses supports that contain lodoacetyl or bromoacetyl groups for the immobilization of ligands through sulfhydryl residues. These supports are usually prepared via the reaction of an amine-containing material with iodoacetic or bromoacetic acid in the presence of ethyldimethylaminopropyl carbodii-mide (EDC) at pH 4 to 5. EDC reacts with the carboxylic acid in iodo- or bromoacetic acid... 

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... 

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. 

Sulfhydryl groups have also been reported to be essential for the action of crystalline, wheat beta-amylase, and titration of the purified enzyme with iodoacetic acid suggested that there were 3.6—3.8 SH groups per molecule. 

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. 

Since exposure to air reconverts the sulfhydryl groups to disulfides, it is necessary to stabilize the reduced forms by alkylation with benzyl chloride or with iodoacetic acid... 

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 ... 

Figure 5.31 ASIB can react with sulfhydryl-containing molecules through its iodoacetate group to form thioether linkages. Subsequent exposure to UV light causes a ring-expansion process to occur, creating a highly reactive dehydroazepine intermediate that can couple to amine-containing molecules.

In another experiment (J ) we treated ozone-resistant and ozone-susceptible varieties of tobacco with toxic doses of oc-iodoacetic acid, and a-iodoacetamide, both sulfhydryl-binding reagents. The symptoms produced by both compounds were similar to those produced by ozone. The severity of the injury also paralleled ozone resistance (Table II). The degree of injury caused by these two compounds also paralleled the ozone susceptibility of leaves of different ages on the same plant. The uppermost, youngest, leaves appear to be most resistant to both the sulfhydryl-binding reagents and to ozone. 

Inhibition by a variety of metal-binding agents competitive with respect to phosphoryl substrates (118-120) has suggested that an enzyme-bound divalent cation (other than Mg2+) may participate also in the binding of phosphate substrates. Observed inhibition by p-chloro-mercuriphenyl sulfonate and iodoacetate suggests the possibility that sulfhydryl groups may also be involved at, or near, the active enzymic site (119, 120). 

Other sulfhydryl reagents, such as p-mercuribenzoate and iodoaceta-mide, produced similar activation (44), except that with these compounds increases in activity were also observed at pH 9.1 (Table I). With p-mercuribenzoate maximum activation was observed when 2-4 sulfhydryl groups were titrated, and with excess reagent catalytic activity was almost completely abolished (44)- Similar results were obtained with FDNB (15). The reactive sulfhydryl groups may be located in apolar regions of the enzyme molecule since they were not affected by N-ethylmaleimide or iodoacetic acid. 

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