Radioiodination procedure

The radioiodination procedure (see Note 9) described here uses IODO-BEADS, which contain the sodium salt of IV-chloro-benzenesulfonamide immobilized on nonporous, polystyrene beads. Immobilization of the oxidizing agent allows for easy separation of the latter from the reaction mixture. This method also prevents the use of reducing agents. 

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. 

Due to the ease of handling and efficiency, the Chloramine-T method is still the most widely used radioiodination procedure and represents an effective way to label a variety of proteins and peptides such as albumins, globulins, neuropeptides, and chemokines. Several other methods, such as the enzymatically catalyzed iodination with lactoperoxidase, have been developed. These methods allow the labeling with mild reaction conditions. In the case of extreme oxidation, sensitive proteins such as pituitary hormones and reactive pre-iodinated compounds such as the Bolton and Hunter reagent can be used to incorporate radioiodine. 

Kabalka, G. W., Mereddy, A. R. 2004. A facile no-carrier-added radioiodination procedure suitable for radiolabeling kits. Nucl. Med. Biol. 31 935-938. 

Using any of the carrier proteins available in highly purified form, eg, TBG or TBPA, a convenient and accurate quantitative determination of and is possible by displacement of radioiodinated or T. This procedure enables their quick determination at low concentrations even in the presence of coundess other substances that occur in body duids (31). In a similar fashion, intact cell nuclei or solubilized proteins from rat fiver cell nuclei, which display high affinities for thyroid hormones, especially T, have been used to establish relative binding affinities of many thyromimetic compounds (7). 

The prevailing procedures for direct coupling of 125I to a protein or other molecule are through the use of oxidizing agents. The in situ preparation of an electrophilic radioiodine... 

Radioiodination of streptavidin uses procedures similar to those used for stable nuclides However, some distinct differences remain, since radioiodinations are... 

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. 

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. 

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. 

Tritium is measured by liquid scintillation counting of a portion of a distilled sample. Several reagents (such as sodium sulphite and silver iodide) can be added in the distillation to prevent interference by radioiodine. The allowed concentration of tritium in water for human consumption is relatively high thus the method presented here is normally adequate for routine determinations. However, if required, lower concentrations of tritium in water can be determined by electrolytic enrichment. The principles of the tritium determination procedure are as follows. 

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. 

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