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

Insulin-like growth factor (IGF) binding to IGF binding proteins is co mmonly assessed by adsorbing free IGF to albumin-coated charcoal and q uantitating bound IGF in the supernatant, but the validity of this tec hnique 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 an d bound I-125-IGF-II. Optimal HPSEC recovery and resolution was obtain ed for IGFBP-1 and IGFBP-6 with low salt buffer (0.1 M sodium phosphat e, pH 7.4), whereas phosphate buffer supplemented with 0.5 M NaCl was optimal for IGFBPs 2-5. Measurement of binding of I-125-IGF-II to IGFB Ps 3-5 using the charcoal assay was also increased by the use of high salt buffer. Under optimal conditions, charcoal measurements of I-125- IGF-II binding to IGFBPs 1-5 were consistently lower than HPSEC measur ements. By competitive binding using unlabeled IGF-II, the binding aff inity 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 d ue to differences in binding capacity. Charcoal adsorbs 15% of cross-l inked I-125-IGF-II:IGFBP complexes which may partially explain the low er binding capacity for IGFBPs 1-5 determined by charcoal adsorption. Charcoal adsorption and HPSEC, therefore, are both valid methods for t he measurement of binding affinities of IGFBPs for IGF-II, but assay c onditions must be validated prior to measurement of binding capacity.