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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.

SUBMITTER: Chandrasekhar S

PROVIDER: S-EPMC4283463 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Thiol-disulfide exchange in peptides derived from human growth hormone.

Chandrasekhar Saradha S Epling Daniel E DE Sophocleous Andreas M AM Topp Elizabeth M EM

Journal of pharmaceutical sciences 20140218 4

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 inves ...[more]

PMID: 24549831

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Project description:Lyophilization (freeze-drying) is frequently used to stabilize protein therapeutics. However, covalent modifications such as thiol-disulfide exchange and disulfide scrambling can occur even in the solid state. The effects of lyophilization and storage of lyophilized powders on the mechanism and kinetics of thiol-disulfide exchange have not been elucidated and are explored here. Reaction kinetics was monitored in peptides corresponding to tryptic fragments of human growth hormone (T20 + T20-T21 or T20 + cT20-T21) during different stages of lyophilization and during storage of the lyophilized powders at 22°C and ambient RH. The concentrations of reactants and products were determined using RP-HPLC and product identity confirmed using liquid chromatography-mass spectrometry. Loss of native disulfide was observed for the reaction of T20 with both linear (T20-T21) and cyclic (cT20-T21) peptides during the primary drying step; however, the native disulfides were regenerated during secondary drying with no further change till the end of lyophilization. Deviations from Arrhenius parameters predicted from solution studies and the absence of buffer effects during lyophilization suggest that factors such as temperature, initial peptide concentration, buffer type, and concentration do not influence thiol-disulfide exchange during lyophilization. Results from a "cold finger" method used to study peptide adsorption to ice indicate that there is no preferential adsorption to the ice surface and that its presence may not influence disulfide reactivity during primary drying. Overall, reaction rates and product distribution differ for the reaction of T20 with T20-T21 or cT20-T21 in the solid state and aqueous solution, whereas the mechanism of thiol-disulfide remains unchanged. Increased reactivity of the cyclic peptide in the solid state suggests that peptide cyclization does not offer protection against lyophilization and that damage induced by a process stress further affects storage stability at 22°C and ambient RH.

Dataset Information

Thiol-disulfide exchange in peptides derived from human growth hormone.

Publications

Thiol-disulfide exchange in peptides derived from human growth hormone.

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