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Binding Properties of Insulin-Like Growth Factor Binding Protein-3 (IGFBP-3), IGFBP-3 N- and C-Terminal Fragments, and Structurally Related Proteins mac25 and Connective Tissue Growth Factor Measured Using a Biosensor

Department of Pediatric Oncology, Otto von Guericke University (P.V., B.H.), Magdeburg 39112, Germany; Department of Pediatrics, Oregon Health Sciences University (Y.O., R.G.R.), Portland, Oregon 97201; and Department of Scientific Computing, Novo Nordisk A/S (R.M.S.), Måløv DK-2760, Denmark

Address all correspondence and requests for reprints to: Dr. Peter Vorwerk, M.D., Department of Pediatric Oncology, Otto von Guericke University, Emanuel Larisch Weg 17-19, D-39112 Magdeburg, Germany. E-mail: . peter.vorwerk{at}medizin.uni-magdeburg.de

We measured the binding of IGF-I and IGF-II to recombinant human N-terminal [residues 1–97; recombinant human IGF-binding protein-3^1–97 (rhIGFBP-3^1–97)] and C-terminal (residues 98–264; rhIGFBP-3^98–264) IGFBP-3 fragments and compared it with IGF binding to intact IGFBP-3 using biosensor analysis. Experiments were carried out in different configurations, either with binding protein or fragment immobilized or with IGF immobilized. These experiments showed that IGF-I and IGF-II bind to IGFBP-3 with affinities of 4–5 x 10⁹ M^-1 and similar binding kinetics. The affinities of both rhIGFBP-3^1–97 and rhIGFBP-3^98–264 for IGF proteins were approximately 3 orders of magnitude less than that of full-length IGFBP-3. These results further support the concept that high affinity binding of IGF to IGF-binding proteins results from a two-site interaction of IGF with both the N- and C-terminal regions of the binding protein.

Binding of insulin to IGFBP-3 and its N- and C-terminal fragments and of IGF-I and IGF-II to the structurally related proteins mac25 and connective tissue growth factor was also investigated. Weak insulin binding to full-length IGFBP-3 could be demonstrated in a few experiments, but we found that binding of IGF-I, IGF-II, and insulin to mac25 or connective tissue growth factor was below the detection limit of the biosensor instrument.

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