Six methods for direct radioiodination of mouse epidermal growth factor compared: effect of nonequivalence in binding behavior between labeled and unlabeled ligand

• Published in: Clinical chemistry (Baltimore, Md.) Vol. 37; no. 10 Pt 1; p. 1749
• Main Authors: Kienhuis, C B, Heuvel, J J, Ross, H A, Swinkels, L M, Foekens, J A, Benraad, T J
• Format: Journal Article
• Language: English
• Published: England 01-10-1991
• Subjects: Animals; Binding, Competitive; Chloramines; Culture Techniques; Epidermal Growth Factor - metabolism; ErbB Receptors - metabolism; Humans; Iodine Radioisotopes; Isotope Labeling; Membranes - metabolism; Mice; Placenta - metabolism; Tosyl Compounds
• ISSN: 0009-9147
• DOI: 10.1093/clinchem/37.10.1749

Mouse epidermal growth factor (EGF) was radioiodinated by six different direct iodination methods. The 125I-labeled EGF preparations were distinguished by analyzing the binding of the radioligand to the EGF receptor (EGFR)-containing human placental membranes. The receptor-binding affinity of EGF labeled with Chloramine T was less than the affinity of unlabeled EGF, which precluded an accurate determination of the specific radioactivity of the 125I-labeled EGF preparation by "self-displacement analysis." Scatchard analysis of competitive binding data (increasing concentrations of unlabeled EGF) obtained with commercially prepared 125I-labeled EGF (Chloramine T method), according to the specific radioactivity stated by the manufacturer, resulted in a substantial underestimation of the apparent number of receptors. Iodination of EGF with Iodogen or Iodo-beads, reagents claimed to be more gentle because of their solid state, also yielded 125I-labeled EGF preparations that were not equivalent to the native EGF in receptor binding. In contrast, equivalence in the ligand-receptor interaction between labeled and unlabeled EGF could be achieved by iodinating EGF with iodine monochloride (ICl), Protag-125, or lactoperoxidase-glucose oxidase-coupled beads (Enzymobeads). Scatchard plots of saturation and competitive binding data obtained with these 125I-labeled EGF preparations produced identical results for apparent receptor number and apparent dissociation constants. Such radioiodinated EGF preparations yield relevant binding data in competition studies of labeled and unlabeled EGF.