Protein Protocols Protein Protocols

The Lactoperoxidase Method 967
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The Lactoperoxidase Method for Radiolabeling Protein
Graham S. Bailey
1. Introduction
This method, introduced by Marchalonis (1), employs lactoperoxidase in the presence
of a trace of hydrogen peroxide to oxidize the radioactive iodide 125 I – to produce
the reactive species 125 I 2 or 125 I + . These reactive species substitute mainly into tyrosine
residues of the protein, although substitution into other amino acid residues can occur
under certain conditions. The oxidation can be stopped by simple dilution. Although
the technique should result in less chance of denaturation of susceptible proteins than
the chloramine T method, it is more technically demanding and is subject to a more
marked variation in optimum reaction conditions.
2. Materials
1. Na125I: 37 MBq (1 mCi) concentration 3.7 GBq/mL (100 mCi/mL).
2. Lactoperoxidase: available from various commercial sources. A stock solution of 10 mg/mL
in 0.1 M sodium acetate buffer, pH 5.6, can be made and stored at –20°C in small aliquots.
A working solution of 20 µg/mL is made by dilution in buffer just prior to use.
3. Buffer A: 0.1 M sodium acetate buffer, pH 5.6 (see Note 1).
4. Buffer B: 0.05 M sodium phosphate buffer containing 0.1% sodium azide, final pH 7.4.
5. Buffer C: 0.05 M sodium phosphate buffer containing 1 M sodium chloride 0.1% bovine
serum albumin and 1% potassium iodide, final pH 7.4.
6. Hydrogen peroxide: A solution of 10 µg/mL is made by dilution just prior to use.
7. Protein to be iodinated: A 0.5–2.5 mg/mL solution is made in buffer A.
It is essential that none of the solutions except buffer B contain sodium azide as
antibacterial agent, since it inhibits lactoperoxidase.
3. Method
1. Into a small plastic test tube (1 × 5.5 cm) are added, in turn the protein to be iodinated
(5 µL), radioactive iodide (5 µL), lactoperoxidase solution (5 µL), and buffer A (45 µL).
2. The reaction is started by the addition of the hydrogen peroxide solution (10 µL) with
mixing (see Note 2).
3. The reaction is stopped after 20 min (see Note 3) by the addition of buffer B (0.5 mL) with
mixing.
4. After 5 min, buffer C (0.5 mL) is added with mixing. The solution is then ready for purification
as described in Chapter 136 (see Note 4).
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
967