Forbes, B.Turner, D.Hodge, S.McNeil, K.Forsberg, G.Wallace, J.2006-07-052006-07-051998Journal of Biological Chemistry, 1998; 273(8):4647-46520021-92581083-351Xhttp://hdl.handle.net/2440/11309We have investigated which region(s) of bovine insulin-like growth factor binding protein-2 (bIGFBP-2) interact with insulin-like growth factors (IGFs) using C-terminally truncated forms of bIGFBP-2. Initially to aid in mutant design, we defined the disulfide bonding pattern of bIGFBP-2 C-terminal region using enzymatic digestion. The pattern is Cys186-Cys220, Cys231-Cys242, and Cys244-Cys265. In addition, cyanogen bromide cleavage of bIGFBP-2 revealed that the N- and C-terminal cysteine-rich domains were not linked by disulfide bonds. Taking the disulfide bonding pattern into consideration, C-terminal truncation mutants were designed and expressed in COS-1 mammalian cells. Following IGF binding assays, a region between residues 222 and 236 was identified as important in IGF binding. Specifically, mutants truncated by 14, 36, and 48 residues from the C terminus bound IGFs to the same extent as wild type (WT) bIGFBP-2. Removal of 63 residues resulted in a greatly reduced (up to 80-fold) ability to bind IGF compared with WT bIGFBP-2. Interestingly this mutant lacked the IGF-II binding preference of WT bIGFBP-2. Residues 236-270 also appeared to play a role in determining IGF binding specificity as their removal resulted in mutants with higher IGF-II binding affinity.enAnimalsCattleDisulfidesTrypsinSomatomedinsInsulin-Like Growth Factor Binding Protein 2Chromatography, High Pressure LiquidPeptide MappingMutagenesisSequence DeletionBinding SitesAmino Acid SequenceMolecular Sequence DataJournal article0030004395001998188210.1074/jbc.273.8.46470000721150000522-s2.0-003254892668401Hodge, S. [0000-0002-3602-9927] [0000-0002-9401-298X]