title: Measurement of insulin-like growth factor (IGF)-II binding to purified IGF binding proteins 1-6: comparison of charcoal adsorption and high performance size exclusion chromatography.
abstract: Insulin-like growth factor (IGF) binding to IGF binding proteins is commonly assessed by adsorbing free IGF to albumin-coated charcoal and quantitating bound IGF in the supernatant, but the validity of this technique has been questioned and many variations have been described. We compared the measurement of binding affinity and capacity of purified IGFBPs 1-6 for IGF-II using charcoal adsorption and Superdex G75 high performance size exclusion chromatography (HPSEC) to separate free and bound 125I-IGF-II. Optimal HPSEC recovery and resolution was obtained for IGFBP-1 and IGFBP-6 with low salt buffer (0.1 M sodium phosphate, pH 7.4), whereas phosphate buffer supplemented with 0.5 M NaCl was optimal for IGFBPs 2-5. Measurement of binding of 125I-IGF-II to IGFBPs 3-5 using the charcoal assay was also increased by the use of high salt buffer. Under optimal conditions, charcoal measurements of 125I-IGF-II binding to IGFBPs 1-5 were consistently lower than HPSEC measurements. By competitive binding using unlabeled IGF-II, the binding affinity of each of the IGFBPs for IGF-II was the same using both methods. Similarly, binding affinities as measured by charcoal assay were not affected by buffer composition. Differences in total binding obtained using the two methods and under different conditions were therefore due to differences in binding capacity. Charcoal adsorbs 15% of cross-linked 125I-IGF-II:IGFBP complexes which may partially explain the lower binding capacity for IGFBPs 1-5 determined by charcoal adsorption. Charcoal adsorption and HPSEC, therefore, are both valid methods for the measurement of binding affinities of IGFBPs for IGF-II, but assay conditions must be validated prior to measurement of binding capacity.
authors.affiliation: Growth and Development Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA. bach@austin.unimelb.edu.au
authors.forename: L A
authors.initials: LA
authors.lastname: Bach
authors.forename: M M
authors.initials: MM
authors.lastname: Rechler
mesh_terms: D000327:Adsorption; D000818:Animals; D001667:Binding, Competitive; D002606:Charcoal; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003432:Cross-Linking Reagents; D006801:Humans; D018969:Insulin-Like Growth Factor Binding Proteins / Q000378:metabolism*; D007335:Insulin-Like Growth Factor II / Q000378:metabolism*; D008722:Methods; D009994:Osmolar Concentration; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins
chemical_list: D003432:Cross-Linking Reagents; D018969:Insulin-Like Growth Factor Binding Proteins; D011994:Recombinant Proteins; D002606:Charcoal; D007335:Insulin-Like Growth Factor II