Sulfhydryls with lodoacetate

The relative reactivity of a-haloacetates toward protein functionalities is sulfhydryl imidazolyl 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 reactivities, but will obviously not create a carboxylate functional group. The acetamide derivatives typically are used only as blocking reagents. 

Dissolve the sulfhydryl-containing protein or macromolecule to be modified at a concentration of 1-10 mg/ml in 50 mM Tris, 0.15 M NaCl, 5 mM 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 0.1 M sodium phosphate, 0.15 M NaCl, 5 mM EDTA, pH 8. 

Add iodacetate to a concentration of 50 mM 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 (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 readjusted, and an aliquot added to the protein solution to give the desired concentration. 

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

An Ellman s assay comparing the unmodified protein to the iodoacetylated protein may be done to assess the degree of modification. 

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