Probing the folding pathways of long R-3 insulin-like growth factor-I (LR(3)IGF-I) and IGF-I via capture and identification of disulfide intermediates by cyanylation methodology and mass spectrometry

This report describes an integrated investigation of the refolding and redu ctive unfolding of insulin-like growth factor (IGF-I) and its variant, long R-3 IGF-I (LR(3)IGF-I), which has a Glu(3) to Arg(3) substitution and a hy drophobic 13-amino acid N-terminal extension. The refolding performed in gl utathione redox buffer was quenched at different time points by adjusting t he pH to 2.0-3.0 with a 1 N HCl solution of 1-cyano-4-dimethylaminopyridini um tetrafluoroborate, which trapped intermediates via cyanylation of free s ulfhydryl groups. The disulfide structure of the intermediates was determin ed by chemical cleavage followed by mass mapping with mass spectrometry. Si x refolding intermediates of IGF-I and three refolding intermediates of LR( 3)IGF-I were isolated and characterized. Folding pathways of IGF-I and LR(3 )IGF-I are proposed based on the time-dependent distribution and disulfide structure of the corresponding trapped intermediates. Similarities and diff erences in the refolding behavior of IGF-I and LR(3)IGF-I are discussed.