Protein Protocols Protein Protocols

964 Bailey
is oxidized by chloramine T in aqueous solution. The oxidation is stopped after a brief
period of time by addition of excess reductant. Unfortunately, some proteins are denatured
under the relatively strong oxidizing conditions, so other methods of radioiodination
that employ more gentle conditions have been devised, e.g., the lactoperoxidase
method (see Chapter 133).
2. Materials
1. Na 125 I: 37 MBq (1 mCi) concentration 3.7 GBq/mL (100 mCi/mL).
2. Buffer A: 0.5 M sodium phosphate buffer, pH 7.4 (see Note 1).
3. Buffer B: 0.05 M sodium phosphate buffer, pH 7.4.
4. Buffer C: 0.01 M sodium phosphate buffer containing 1 M sodium chloride, 0.1% bovine
serum albumin, and 1% potassium iodide, final pH 7.4.
5. Chloramine T solution: A 2 mg/mL solution in buffer B is made just prior to use (see Note 2).
6. Reductant: A 1 mg/mL solution of sodium metabisulfite in buffer C is made just prior to use.
7. Protein to be iodinated: A 0.5–2.5 mg/mL solution is made in buffer B.
3. Method
1. Into a small plastic test tube (1 × 5.5 cm) are added successively the protein to be iodinated
(10 µL), radioactive iodide (5 µL), buffer A (50 µL), and chloramine T solution (25 µL)
(see Notes 3 and 4).
2. After mixing by gentle shaking, the solution is allowed to stand for 30 s to allow
radioiodination to take place (see Note 5).
3. Sodium metabisulfite solution (500 µL) is added to stop the radioiodination, and the
resultant solution is mixed. It is then ready for purification as described in Chapter 136
and Note 6.
4. Notes
1. The pH optimum for the iodination of tyrosine residues of a protein by this method is
about pH 7.4. Lower yields of iodinated protein are obtained at pH values below about 6.5
and above about 8.5. Indeed, above pH 8.5 the iodination of histidine residues appears to
be favored.
2. If the protein is seriously damaged by the use of 50 µg of chloramine T, it may be worthwhile
repeating the radioiodination using much less oxidant (10 µg or less). Obviously,
the minimum amount of chloramine T that can be used will depend, among other factors,
on the nature and amount of protein to be iodinated.
3. The total volume of the reaction should be as low as practically possible to achieve a rapid
and efficient incorporation of the radioactive iodine into the protein.
4. It is normal to carry out the method at room temperature. However, if the protein is especially
labile, it may be beneficial to run the procedure at a lower temperature and for a
longer period of time.
5. Because of the small volumes of reactants that are employed, it is essential to ensure
adequate mixing at the outset of the reaction. Inadequate mixing is one of the most common
reasons for a poor yield of radioiodinated protein by this procedure.
6. It is possible to carry out this type of reaction using an insoluble derivative of the sodium
salt of N-chloro-benzene sulfonamide as the oxidant. The insoluble oxidant is available
commercially (Iodo-Beads, Pierce, Rockford, IL). It offers a number of advantages over
the employment of soluble chloramine T. It produces a lower risk of oxidative damage to
the protein, and the reaction is stopped simply by removing the beads from the reaction
mixture, thus avoiding any damage caused by the reductant.