Amines with 2-Iminothiolane Trauts Reagent

At high pH (10), Traut s reagent also is reactive with aliphatic and aromatic hydroxyl groups, although the rate of reaction with these groups is only about O.Ol that of primary amines. In the absence of amines, however, carbohydrates such as agarose or cellulose membranes can be modified to contain sulfhydryl residues (Alagon and 

Polysaccharides modified in this manner are effective in covalently cross-linking antibodies for use in immunoassay procedures. 

Proteins modified with 2-iminothiolane are subject to disulfide formation on sulf-hydryl oxidation. This can cause unwanted conjugation, potentially precipitating the protein. The addition of a metal chelating agent such as EDTA (0.01—0.1 M) will prevent metal-catalyzed oxidation and maintain sulfhydryl stability. In the presence of some serum proteins (i.e., BSA) a 0.1 M concentration of EDTA may be necessary to prevent metal-catalyzed oxidation, presumably due to the high contamination of iron from hemolyzed blood. 

Prepare the protein or macromolecule to be thiolated in a non-amine-containing buffer at pH 8.0. For the modification of ribosomal proteins (often cited in the literature) use 50 mM triethanolamine hydrochloride, 1 mM MgCl2, 50 mM KCl, pH 8. The magnesium and potassium salts are for stabilization of some ribosomal proteins. If other proteins are to be thiolated, the same buffer may be used without added salts for stabilization. Alternatively, 50 mM sodium phosphate, 0.15 M NaCl, pH 8, or 0.1 M sodium borate, pH 8.0 may be used. For the modification of polysaccharides, use 20 mM sodium borax, pH 10, to produce reactivity toward carbohydrate hydroxyl residues. Dissolve the protein to be modified at a concentration of 10 mg/ml in the reaction buffer of choice. Lower concentrations also may be used with a proportional scaling back of added 2-iminothiolane. 

Dissolve the Traut s reagent (Pierce) in water at a concentration of 2 mg/ml (makes a 14.5 mM stock solution). The solution should be used immediately. 

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Traut s reagent is fully water-soluble and reacts with primary amines in the pH range 7—10. The cyclic imidothioester is stable to hydrolysis at acid pH values, but its half-life in solution decreases as the pH increases beyond neutrality. However, even at pH 8 in 25 mM triethanolamine the rate of sulfhydryl formation without added primary amine was found to be negligible. On addition of dipeptide amine, the reagent reacted quickly as evidenced by the production of Ellman s reagent color. The rate of reaction also can be followed by 2-iminothiolane s absorbance at 248 nm (Xmax e = 8 840 M 1 cm 1). As the cyclic imidate reacts with amines, its absorbance at... 

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