title: Putative folding pathway of insulin-like growth factor-I.
abstract: Insulin-like growth factor-I (IGF-I) has three disulfide bonds and refolding of the fully reduced molecule generates varying ratios of correctly (PII) and incorrectly (PI) folded forms via several intermediates. All of the intermediates have the disulfide bond between Cys18 and 61 formed, indicating that formation of this disulfide is the first step in refolding. In order to further understand the refolding pathway, two intermediate froms, PIII with the additional disulfide Cys(6/47) formed and PIIIa with Cys(6/48) formed, were isolated. The oxidation of the remaining Cys48 and 52 in PIII and Cys47 and 52 in PIIIa would lead to PI and PII, respectively; however, air oxidation of these resulted in a rapid reshuffling into other intermediates as well as folding into the fully oxidized forms, and this occurred whether refolding was started with PIII or PIIIa. When oxidation occurred in the presence of an excess of oxidized glutathione, the predominant species generated were various glutathione adducts regardless of the initial intermediate form, indicating that formation of the last disulfide bond is not a favorable process relative to disulfide exchange when excess disulfides from oxidized glutathione are present. Interestingly, if 80 microM copper sulfate, an oxidant, is added to the refolding buffer, PIII resulted in formation of the PI form alone, whereas PIIIa resulted in the PII form alone. It was concluded from these results that the intermediate forms of IGF-1 can rapidly reshuffle between different disulfide structures, and that formation of the last disulfide bond is not as favorable a process as the conversion to other intermediates. The oxidation to form the last disulfide bond in PIII or PIIIa is accelerated and hence the interconversion to other intermediates is kinetically minimized only in the presence of copper sulfate. It appears, therefore, that the two intermediate forms, PIII and PIIIa, are the precursors of the corresponding fully oxidized forms, but their conversions are not energetically a favorable process.
authors.affiliation: Amgen Inc., Thousand Oaks, California 91320, USA.
authors.forename: R D
authors.initials: RD
authors.lastname: Rosenfeld
authors.forename: J A
authors.initials: JA
authors.lastname: Miller
authors.forename: L O
authors.initials: LO
authors.lastname: Narhi
authors.forename: N
authors.initials: N
authors.lastname: Hawkins
authors.forename: V
authors.initials: V
authors.lastname: Katta
authors.forename: S
authors.initials: S
authors.lastname: Lauren
authors.forename: M A
authors.initials: MA
authors.lastname: Weiss
authors.forename: T
authors.initials: T
authors.lastname: Arakawa
mesh_terms: D002851:Chromatography, High Pressure Liquid; D019327:Copper Sulfate / Q000494:pharmacology; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D019803:Glutathione Disulfide; D007334:Insulin-Like Growth Factor I / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D017510:Protein Folding; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism; D014269:Trifluoroacetic Acid / Q000494:pharmacology
chemical_list: D004220:Disulfides; D013438:Sulfhydryl Compounds; D007334:Insulin-Like Growth Factor I; D014269:Trifluoroacetic Acid; D005978:Glutathione; D019327:Copper Sulfate; D019803:Glutathione Disulfide