title: Thiol-disulfide exchange in peptides derived from human growth hormone.
abstract: Disulfide bonds stabilize proteins by cross-linking distant regions into a compact three-dimensional structure. They can also participate in hydrolytic and oxidative pathways to form nonnative disulfide bonds and other reactive species. Such covalent modifications can contribute to protein aggregation. Here, we present experimental data for the mechanism of thiol-disulfide exchange in tryptic peptides derived from human growth hormone in aqueous solution. Reaction kinetics was monitored to investigate the effect of pH (6.0-10.0), temperature (4-50°C), oxidation suppressants [ethylenediaminetetraacetic acid (EDTA) and N2 sparging], and peptide secondary structure (amide cyclized vs. open form). The concentrations of free thiol containing peptides, scrambled disulfides, and native disulfide-linked peptides generated via thiol-disulfide exchange and oxidation reactions were determined using reverse-phase HPLC and liquid chromatography-mass spectrometry. Concentration versus time data were fitted to a mathematical model using nonlinear least squares regression analysis. At all pH values, the model was able to fit the data with R(2) ≥ 0.95. Excluding oxidation suppressants (EDTA and N2 sparging) resulted in an increase in the formation of scrambled disulfides via oxidative pathways but did not influence the intrinsic rate of thiol-disulfide exchange. In addition, peptide secondary structure was found to influence the rate of thiol-disulfide exchange.
authors.affiliation: Department of Industrial and Physical Pharmacy, Purdue University, West Lafayette, IN.
authors.forename: Saradha
authors.initials: S
authors.lastname: Chandrasekhar
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Epling
authors.forename: Andreas M
authors.initials: AM
authors.lastname: Sophocleous
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Topp
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry*; D004492:Edetic Acid / Q000737:chemistry; D013006:Growth Hormone / Q000737:chemistry*; D019382:Human Growth Hormone / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D013438:Sulfhydryl Compounds / Q000737:chemistry*
chemical_list: D004220:Disulfides; D013438:Sulfhydryl Compounds; D019382:Human Growth Hormone; D013006:Growth Hormone; D004492:Edetic Acid
keywords: aggregation; human growth hormone; kinetics; liquid chromatography; oxidation; peptides; proteins; thiol-disulfide exchange