Chloramine-T, radioiodination

Below are listed the basic steps of the modified method of chloramine-T radioiodination of proteins that have evolved in our laboratory over the past 15 years. We routinely radioiodinate 20-30 proteins at a time using this method. The entire radioiodination procedure is conducted in an ice bath and can be accomplished in about 60 min. 

Chloramine-T radioiodination of proteins involves both oxidation of radioiodide to its reactive state, H2OD, and oxidation of sulfhydryl groups of the protein molecules. There must be sufficient chloramine-T... 

Chloramine-T, or N-chlorotoluenesulfonamide, has been one of the most popular oxidizing reagents used for radioiodination techniques since its introduction by Greenwood et al. in 1963 (Sigma). It has strong oxidizing properties that readily lead to the formation of the required... 

Lee, D.S.C., and Criffiths, B.W. (1984) Comparative studies of Iodo-Bead and chloramine-T methods for the radioiodination of human alpha-fetoprotein./. Immunol. Meth. 74, 181-189. 

The most common method of radiolabeling employs Chloramine-T, the sodium salt of the N-monochloro derivative of p-toluenesulfonamide (Fig. 17). It breaks down slowly in aqueous solution to hypochlorous acid and is used as a mild oxidizing agent in radioiodination reactions. Other oxidation reagents used in radioiodination include... 

Several applications of photoreactive peptides require the presence of a radionuclide to allow specific and sensitive detection of the photo-cross-linked conjugates. In several cases, radioiodination of tyrosyl moieties and radiolabeled Bolton-Hunter reagents have been used. However, the presence of a radiolabel within the benzophenone photophore is desirable, particularly when the objective is to identify the site of photo-insertion of benzophenone. To this end some radiolabeled, benzophenone-based compounds have been developed and used in peptide synthesis, in particular tritiated Phe(4-Bz) (Scheme 24)J2161 [1-14C-carboxy]-4-benzoylbenzoic acid,1221 and 4-benzoyl-(2,3-3H2)-dihydrocinnamic acid.[154l In addition, 4-(4-hydroxybenzoyl)phenylalanine (Scheme 25) has been directly radioiodinated with Na125I and Chloramine-T)151 ... 

IODO-GEN (Pierce), first described by Fraker and Speck in 1978, is 1,3,4,6-tetrachloro-3a,6a-diphenylglycouril, an N-haloamine derivative with oxidizing properties similar to those of IODO-BEADS and chloramine-T. The compound is insoluble in aqueous solution, therefore making it a type of solid-phase radioiodination reagent. However, unlike IODO-BEADS, wherein the oxidizing group is immobilized on an-... 

Radioiodination of galactose oxidase. The chloramine T procedure (12) was used for the radioiodination of galactose oxidase. The enzyme, solubilized in 0.01 M sodium phosphate buffer, pH 7.4, was labeled using 1 mCi Na 125I (13-17 mCi/ngl), 0.42 mM chloramine T (Eastman), and 1.14 mM sodium metabisulfite (Baker). Unreacted iodide was separated from the iodination enzyme by dialysis, and the enzyme was diluted in 0.1 M sodium phosphate buffer, pH 7.4, containing 0.001 M cupric sulfate. 

Radioiodination of vinylboronic acid derivatives has been applied443-445 for the synthesis of 17 tf-iodovinylestradiol derivatives443 (equations 206) and (E)-1 -[123I]iododecene, 306 (equation 207). In the iodination of a vinylboronic acid, chloramine-T has been found to be the most efficient oxidant under both carrier-added and NCA conditions and has been applied in reaction 207. 

Initiate radioiodination by adding freshly prepared chloramine-T. Chloramine-T is usually prepared at a concentration of 500 / g/ml in 0.1 M phosphate buffer, pH 7. The ratio of chloramine-T to protein most commonly used is 10 /u.g per milligram of protein. 

No laboratory procedure is so simple and direct that there are no areas of potential error. The following outlines the most common problems we have experienced with radioiodination by chloramine-T and some of the measures taken to increase the usefulness of radioiodinated proteins. 

Efficiency of radioiodination is enhanced by minimizing the volume of reactants. In our laboratory the efficiency of radioiodination averages 50% when the following conditions are met (a) protein at 0.5 mg in volumes from 0.5 to 2 ml (b) chloramine-T at 100-500 jxg/ml, 10 fig of chloramine-T added per milligram of protein in volumes from 10 to 50 /il (c) neutralized radioiodine from 50 to 5000 /xCi in volumes less than 100 fi for a final total volume of approximately 1-2 ml. 

Partial List of Proteins Radioiodinated by the Chloramine-T Method... 

There are some proteins that can be radioiodinated successfully by chloramine-T but in the process lose some of their biological activity. The first five complement components fall in this category. Alternative methods of radioiodination must be used in such cases, as reported in this volume [12]. A partial list of proteins radioiodinated with chloramine-T without detectable loss of immunologic or enzymic activity can be found in the table. We have not been able to achieve greater than 10% incorporation of radioiodine into ovalbumin using chloramine-T at increased ratios of chloramine-T to ovalbumin. 

Some areas of investigation are restricted by the requirement for purified proteins because these may be limited in quantity of source material and their purification often involves tedious and inefficient procedures. Elder et al. have reported a unique approach that bypasses some of these restrictions in their studies of viral and plasma membrane proteins. After separation of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, they remove the gel slice containing the protein band of interest and radioiodinate it by chloramine-T while still in the gel. This application should prove to be useful in other systems. 

The labelling of TATE (AnaSpec) and DOTATATE (piCHEM R D) with [ T]NaI (Nordion) was optimized using the chloramine T method [3.1]. A solution of 10 jxg of peptide in 40 pL of phosphate buffer solution (PBS) (O.IM, pH7.5) was transferred to a reaction vial. After addition of the chloramine T solution (5 jxg/5 LxL) and 5-10 pL of radioiodine solution (37-111 MBq), the vial was carefully vortexed and the reaction was allowed to proceed for 1-3 min at room temperature. The reaction was terminated by addition of the sodium metabisulfite solution (10 pg/5 pL). Studies were carried out by varying the molar ratios of the peptide, the DOTATATE and the radionuclide. Labelling procedures with high activity [ I]NaI were also evaluated employing 1110 and 2775 MBq (30 and 75 mCi), 30 and 100 pg of the peptide, 50 and 100 pg of chloramine T and metabisulfite, respectively. The stability of these preparations was evaluated for 48 h. 

Radiolabelling with 1 or 1 was carried out by the chloramine T method, and radioiodinated analogues were easily isolated with high chemical and radiochemical purity by HPLC, as shown for the labelling reaction mixture of I-Sugar-TATE in the representative chromatogram in Fig. 6.4. 

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