Oxidative radioiodination damage to human lactoferrin.
Oxidative iodination of human lactoferrin (Lf) as commonly performed by using the chloramine-T, the Iodogen or the lactoperoxidase method produces an unreliable tracer protein because of excessive and heterogeneous polymer formation. Before iodination a minor tetramer fraction may be demonstrable in iron-saturated Lf only. Iodination-induced polymerization of iron-poor as well as iron-saturated Lf occurs independently of the presence or absence of 10 mM-EDTA and the 125I-/Lf molar ratio used for iodination. 125I-Lf polymers are mainly covalently linked, as suggested by the lack of substantial dissociation in SDS/polyacrylamide-gel electrophoresis. Damage to the 125I-Lf monomer may be another consequence of oxidative iodination. This is demonstrated in SDS/polyacrylamide-gel electrophoresis where 50% of the radioactivity of apparently normal monomer (Mr 75,000) is displaced to a lower-Mr region (30,000-67,000) after reduction with dithiothreitol. Non-oxidative iodination by the Bolton-Hunter technique produces an antigenetically stable tracer that is not being subjected to polymerization and monomer degradation as judged by high-performance gel chromatography and SDS/polyacrylamide-gel electrophoresis with and without dithiothreitol treatment. It is concluded that oxidation in itself leads to covalent non-disulphide cross-linking between human Lf molecules and, possibly, to intramolecular peptide-bond breaking becoming unmasked under reducing conditions. In biological experiments with human 125I-Lf this problem should be carefully considered.
D000915:Antibody Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007457:Iodine Radioisotopes; D007501:Iron / Q000378:metabolism; D007781:Lactoferrin / Q000276:immunology / Q000528:radiation effects*; D007782:Lactoglobulins / Q000528:radiation effects*; D046911:Macromolecular Substances; D010084:Oxidation-Reduction
D007457:Iodine Radioisotopes; D007782:Lactoglobulins; D046911:Macromolecular Substances; D007501:Iron; D007781:Lactoferrin