Six methods for direct radioiodination of mouse epidermal growth factor compared: effect of nonequivalence in binding behavior between labeled and unlabeled ligand.
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.
Department of Experimental and Chemical Endocrinology, University Hospital Nijmegen, The Netherlands.
C B
CB
Kienhuis
J J
JJ
Heuvel
H A
HA
Ross
L M
LM
Swinkels
J A
JA
Foekens
T J
TJ
Benraad
D000818:Animals; D001667:Binding, Competitive; D002700:Chloramines; D046508:Culture Techniques; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D007553:Isotope Labeling; D008566:Membranes / Q000378:metabolism; D051379:Mice; D010920:Placenta / Q000378:metabolism; D014105:Tosyl Compounds
D002700:Chloramines; D007457:Iodine Radioisotopes; D014105:Tosyl Compounds; C016300:chloramine-T; D004815:Epidermal Growth Factor; D066246:ErbB Receptors