Characterization of the protein which binds insulin‐like growth factor in human serum

The binding of the 125 I‐labelled insulin‐like growth factors I and II ( l25 I‐IGF I and 125 I‐IGF II) to the high‐molecular‐mass binding protein of human serum was characterized. With diluted human serum both growth factors showed optimal specific binding at 4°C and pH 5–6. When 0.1% Triton X‐100 was present in the incubation buffer an increase in the affinity of the IGF‐binding protein was induced, which produced an enhanced binding of IGF I and IGF II. Competition experiments with various peptide hormones revealed that the native IGF‐binding protein complex binds both the IGF I and IGF II with high specificity. Analysis of binding data according to the method of Scatchard resulted in linear plots for IGF I and IGF II respectively, indicating that in human serum only a single class of non‐interacting binding sites is present. At optimal binding conditions the dissociation constants were determined to be 0.28 × 10 −9 M for IGF I binding and 0.66 × 10 −9 M for IGF II. Human serum was gel‐filtered on Sepharose CL‐6B at neutral pH and the eluate was assayed for binding activity with both IGF I and IGF II. One peak with an apparent molecular mass of 175 kDa and a Stokes radius of 4.8 nm was determined for both growth factors. Thus, our data suggest that human serum contains one class of high‐molecular‐mass binding protein with comparable binding characteristics for IGF I and IGF II.