| Autor: |
Nairn NW; Allozyne , Seattle, Washington 98102, United States., Bariola PA; Allozyne , Seattle, Washington 98102, United States., Graddis TJ; Allozyne , Seattle, Washington 98102, United States., VanBrunt MP; Allozyne , Seattle, Washington 98102, United States., Wang A; Allozyne , Seattle, Washington 98102, United States., Li G; Allozyne , Seattle, Washington 98102, United States., Grabstein K; Allozyne , Seattle, Washington 98102, United States. |
| Abstrakt: |
Bioconjugation by copper-catalyzed azide-alkyne cycloaddition (CuAAC) provides a powerful means to produce site-specifically modified proteins. However, the use of a copper catalyst brings about the possible generation of reactive oxygen species that could cause degradation of vulnerable amino acid residues. We investigated whether PEGylation by CuAAC caused any modifications to the therapeutic protein interferon beta-1b, which was produced via global amino acid substitution with azidohomo-alanine at the N-terminus and contains no methionine residues. Using previously reported reaction conditions, LC-MS peptide mapping detected +32 Da and +48 Da oxidation modifications of tryptic peptides 28-33 (LEYCLK) and 137-147 (EYSHCAWTIVR) in the protein post-PEGylation. The oxidative degradation increased with reaction time, whereas reducing the copper concentration slowed the PEGylation rate as well as the oxidation rate. Replacing dithiothreitol (DTT) with any of five different monothiol reducing agents in anaerobic conditions allowed efficient PEGylation in 2-4 h and abrogated oxidative degradation. Free cysteine provided reproducible reaction results as a reducing agent in this system and has been successfully applied to other protein conjugations. Monothiol reducing agents, such as cysteine, may be useful tools as protective reducing agents for CuAAC in some bioconjugation systems. |
