Protein Protocols Protein Protocols

Radioiodination Using IODO-GEN 971
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Preparation of 125 I-Labeled Peptides and Proteins
with High Specific Activity Using IODO-GEN
J. Michael Conlon
1. Introduction
The reagent IODO-GEN (1,3,4, 6-tetrachloro-3α,6α-diphenylglycoluril) was first
introduced by Fraker and Speck in 1978 (1) and rapidly found widespread use for the
radioiodination of both peptides and proteins (2). Although the usual application of the
reagent in the laboratory is for introduction of an atom of 125I, IODO-GEN has been
used successfully for the preparation of proteins, particularly monoclonal antibodies,
labeled with 131I and 123I for use in immunoscintigraphy and positron emission tomography
(3–5). This chapter addresses only the use of IODO-GEN to radiolabel pure
peptides/proteins in aqueous solution, but iodination of proteins on the cell surface of a
wide range of intact eukaryotic and prokaryotic cells (6,7) and on subcellular organelles (8)
has been accomplished using the reagent. The advantages of IODO-GEN over alternative
reagents previously used for radiolabeling such as chloramine T, lactoperoxidase/
hydrogen peroxide, and 125I-labeled Bolton–Hunter reagent (N-succinimidyl-3
[4-hydroxyphenyl] propionate) are that the reaction is rapid, technically simple to perform,
and gives reproducibly high levels of incorporation of radioactivity with minimal
oxidative damage to the protein.
The structure of IODO-GEN is shown in Fig. 1. The reagent is virtually insoluble in
water and so a solution in dichloromethane or chloroform is used to prepare a thin film
of material on the walls of the reaction vessel. Addition of an aqueous solution of Na +
125I – generates the reactive intermediate (I + /I3 + ) that participates in electrophilic attack
primarily at tyrosine but also at histidine residues in the peptide/protein. Reaction is
terminated simply by transferring the contents of the reaction vessel to a clean tube,
thereby obviating the need to add a reducing agent. In comparison to other radioiodination
methods, particularly the use of chloramine T and lactoperoxidase, oxidative
damage to sensitive residues in the peptide/protein (particularly methionine and
tryptophan) is much less using IODO-GEN (9). To minimize oxidative damage even
further and to limit production of diiodotyrosyl derivatives, the strategy of trace labeling
is recommended. This involves radioiodination of only approx 10% of the molecules
and necessitates the separation, as completely as possible, of the radiolabeled
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
971