Thiol-disulfide exchange in peptides derived from human growth hormone.
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.
['Chandrasekhar|Saradha|S|', 'Epling|Daniel E|DE|', 'Sophocleous|Andreas M|AM|', 'Topp|Elizabeth M|EM|']
D000595:Amino Acid Sequence D002851:Chromatography, High Pressure Liquid D004220:Disulfides D004492:Edetic Acid D013006:Growth Hormone D019382:Human Growth Hormone D006801:Humans D006863:Hydrogen-Ion Concentration D007700:Kinetics D010084:Oxidation-Reduction D017433:Protein Structure, Secondary D013438:Sulfhydryl Compounds
thiol-disulfide exchange peptides human growth hormone proteins oxidation aggregation kinetics liquid chromatography