Radioiodination of sulfhydryl-sensitive proteins.

Radioiodination with rz51 has emerged as the method of choice for labeling protein to high specific radioactivity. A number of procedures have been developed to iodinate proteins under mild conditions and with reproducible results (l-7). Of these techniques, none is suitable for the iodination of proteins having thiol groups. Exposure to the oxidants Nchloro-p-toluene sulfonamide (chloramine-T) (l), hydrogen peroxide (2-4), iodinemonochloride (5), and iodine itself (7) may result in oxidation of sulfhydryl groups with the concomitant disruption of tertiary structure or the chemical modification of a specific amino acid residue in the active site. An alternative method involves the acylation of protein with N-succinimidyl-3-(3-[1251]iodo-4-hydroxyphenyl) propionate (6). However, this active ester could react with cysteinyl as well as lysyl residues and thus also disturb the structural integrity of the protein. The present report describes a simple, rapid, and reproducible technique for the radioiodination of sulfhydryl-containing proteins to high specific activity. Aniline is iodinated with Na[1251] in the presence of an oxidant, the product separated by solvent extraction, diazotized, and coupled to the protein. This method which was first suggested by Boyd et al. (8) has been applied successfully to the iodination of three proteins known to require cysteinyl residues for full activity.