High-performance liquid chromatography of 125I-labeled mouse epidermal growth factor radioiodinated by six different methods.
Six different procedures for radioiodination of mouse epidermal growth factor (EGF) all resulted in a heterogeneous 125I-labeled EGF preparation, as analyzed by reversed-phase HPLC. EGF preparations that had been iodinated with Chloramine T, lodogen, or lodo-beads were found mainly to consist of oxidized 125I-labeled EGF moieties. In contrast, the heterogeneous 125I-labeled EGF preparations obtained by using iodine monochloride, Protag-125, or lactoperoxidase-glucose oxidase-coupled beads (Enzymobeads) contained insignificant amounts of oxidized EGF entities. Ligand equivalence analysis (LEA) of distinct HPLC column fractions, obtained after preparative separation of Chloramine T-125I-labeled EGF, showed that the receptor-binding affinity of the tracer in all subfractions was less than the affinity of unlabeled EGF. This implies that HPLC purification of these 125I-labeled EGF preparations does not yield 125I-labeled EGF preparations with ligand equivalence. However, all but one HPLC column fraction of Enzymobeads-125I-labeled EGF showed ligand equivalence. Despite the small amount of the nonequivalent component in the Enzymobeads-labeled tracer, the nonchromatographed 125I-labeled EGF preparation showed ligand equivalence. No significant differences were observed in the maximal binding capacity of the different 125I-labeled EGF preparations.
Kienhuis
C B
CB
Department of Experimental and Chemical Endocrinology, University Hospital Nijmegen, The Netherlands.
Heuvel
J J
JJ
Ross
H A
HA
Foekens
J A
JA
Benraad
T J
TJ
D000818:Animals; D002700:Chloramines; D002851:Chromatography, High Pressure Liquid; D004815:Epidermal Growth Factor / Q000302:isolation & purification*; D006861:Hydrogen Peroxide; D007457:Iodine Radioisotopes; D007553:Isotope Labeling / Q000379:methods*; D051379:Mice; D008863:Microspheres; D010084:Oxidation-Reduction; D014105:Tosyl Compounds; D014508:Urea / Q000031:analogs & derivatives
D002700:Chloramines; D007457:Iodine Radioisotopes; D014105:Tosyl Compounds; C016300:chloramine-T; C012385:1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenylglycoluril; D004815:Epidermal Growth Factor; D014508:Urea; D006861:Hydrogen Peroxide