Effect of macromolecular crowding on protein oxidation: Consequences on the rate, extent and oxidation pathways
Research output: Contribution to journal › Journal article › Research › peer-review
- Effect of macromolecular crowding
Final published version, 6.02 MB, PDF document
Biological systems are heterogeneous and crowded environments. Such packed milieus are expected to modulate reactions both inside and outside the cell, including protein oxidation. In this work, we explored the effect of macromolecular crowding on the rate and extent of oxidation of Trp and Tyr, in free amino acids, peptides and proteins. These species were chosen as they are readily oxidized and contribute to damage propagation. Dextran was employed as an inert crowding agent, as this polymer decreases the fraction of volume available to other (macro)molecules. Kinetic analysis demonstrated that dextran enhanced the rate of oxidation of free Trp, and peptide Trp, elicited by AAPH-derived peroxyl radicals. For free Trp, the rates of oxidation were 15.0 ± 2.1 and 30.5 ± 3.4 μM min−1 without and with dextran (60 mg mL−1) respectively. Significant increases were also detected for peptide-incorporated Trp. Dextran increased the extent of Trp consumption (up to 2-fold) and induced short chain reactions. In contrast, Tyr oxidation was not affected by the presence of dextran. Studies on proteins, using SDS-PAGE and LC-MS, indicated that oxidation was also affected by crowding, with enhanced amino acid loss (45% for casein), chain reactions and altered extents of oligomer formation. The overall effects of dextran-mediated crowding were however dependent on the protein structure. Overall, these data indicate that molecular crowding, as commonly encountered in biological systems affect the rates, and extents of oxidation, and particularly of Trp residues, illustrating the importance of appropriate choice of in vitro systems to study biological oxidations.
|Number of pages||15|
|Publication status||Published - 2021|
- Chain reactions, Macromolecular crowding, Peroxyl radicals, Protein oxidation, Tryptophan, Tyrosine