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Volume 271, Number 24, Issue of June 14, 1996 pp. 13948-13952
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Biosensor Measurement of the Binding of Insulin-like Growth Factors I and II and Their Analogues to the Insulin-like Growth Factor-binding Protein-3

(Received for publication, February 15, 1996)

Anders Heding , Raj Gill ^§ , Yasushi Ogawa ^¶ , Pierre De Meyts and Ronald M. Shymko

From the  Hagedorn Research Institute, Niels Steensens Vej 6, DK-2820 Gentofte, Denmark, the ^§ Department of Crystallography, Birkbeck College, Malet Street, London WC1E 7HX, United Kingdom, and ^¶ Celtrix Pharmaceuticals, Inc., Santa Clara, California 95052

Most insulin-like growth factor (IGF) molecules in the circulation are found in a 150-kDa complex containing IGF-binding protein-3 (IGFBP-3) and an acid-labile subunit, which does not itself bind IGF. Affinities (K_d values) between 0.03 and 0.5 nM have been reported for IGF-I/IGFBP-3 binding, but no kinetic data are available. In this study we measured the high affinity binding of unlabeled IGFs and IGF analogues to recombinant unglycosylated IGFBP-3, using a BIAcore^TM instrument (Pharmacia Biosensor AB). IGF-I binding showed fast association and slow non-first-order dissociation kinetics, and an equilibrium K_d of 0.23 nM. IGF-II had similar kinetics with slightly higher affinity. Analogues with mutations in the first 3 amino acids of the B-region (des(, , ) IGF-I and long IGF-I) showed 25 and 50 times lower affinity than IGF-I. Replacement of residues 28-37 by Gly-Gly-Gly-Gly or deletion of residues 29-41 in the C-region had little effect on the kinetic parameters, contrasting with the markedly impaired binding of these analogues to the IGF-I receptor. Swapping of the disulfide bridges in IGF-I and the C-region mutants decreased the affinity dramatically for IGFBP-3, primarily by decreasing the association rate. Insulin had approximately 1000 times lower affinity than IGF-I.

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