THE INSULIN-LIKE GROWTH-FACTOR (IGF) BINDING-SITE OF BOVINE INSULIN-LIKE GROWTH-FACTOR BINDING PROTEIN-2 (BIGFBP-2) PROBED BY IODINATION

The insulin-like growth factor (IGF) binding site of bovine insulin-li ke growth factor binding protein 2 (bIGFBP-2) has been probed by chemi cal iodination. Tyrosyl residues of bIGFBP-2 were reacted by chloramin e T-mediated iodination. The modification patterns of free bIGFBP-2 an d bIGFBP-2 associated with insulin-like growth factor II (IGF-II) were compared by tryptic mapping using electrospray mass spectrometry and N-terminal sequencing. The presence of bound IGF-II resulted in protec tion of tyrosine at position 60 from iodination measured by the relati ve loss of tyrosine specific fluorescence and the incorporation of the radioisotope I-125. In addition, the pattern of iodine incorporation of bIGFBP-2 was not different whether IGF-I or IGF-II was the protecti ve ligand. bIGFBP-2, when iodinated alone sustained a 8-fold loss of b inding affinity for IGF-I and a 4-fold loss in binding affinity for IG F-II. In contrast, bIGFBP-2 iodinated while complexed with either IGF- I or IGF-II retained the same binding affinity for IGF-I or IGF-II as non-iodinated bIGFBP-2. We conclude that tyrosine 60 Lies either in a region of bIGFBP-2 which directly interacts with both IGF-I and IGF-II or lies in a region of bIGFBP-2 which undergoes a conformational chan ge that is important for IGF binding. Furthermore, iodination of tyros ine residues at positions 71, 98, 213, 226, and 269 has no detectable impact on binding of bIGFBP-2 to the IGFs.