Cold chemical oxidation of proteins

Abstract

Various methods of protein footprinting use hydrogen peroxide as an oxidant. Its removal by various solid-phase desalting methods, catalase treatment, or freeze drying after the footprinting is critical to ensure no uncontrolled oxidation. Although catalase treatment removes hydrogen peroxide with little loss of protein or additional protein oxidation, we discovered that freeze drying or freezing of the protein in a peroxide solution does lead to protein oxidation. Interestingly, the oxidation is not a result of freeze or thaw processes but is dependent on the temperature and length of time for incubation. After 2 h, apomyoglobin undergoes almost-complete single oxidation at -80 degrees C and double oxidation at -15 degrees C. Minimal oxidation is observed at 4 and 22 degrees C, compared to oxidation at -80 or -15 degrees C. The concentration of hydrogen peroxide is critical; 75 mM (0.2%) is required to oxidize >50% of the protein at -15 degrees C and 100 mM (0.3%) is required at -80 degrees C. In addition to Met, approximately 5% of the tryptophan and tyrosine residues are oxidized, as well as lower amounts of His and Phe. Oxidation of Val 68 and Val 17 (a buried residue) also occurs, with the oxidation of Val 17 likely occurring by electron transfer from one of two of the oxidized aromatic residues that are in contact with Val 17. Here, we describe the need to remove the hydrogen peroxide prior to cold storage of proteins, and we also report some preliminary results pertaining to the mechanism of cold, solid-state oxidation.

Figure 1

Mass spectra of 20 μM apomyoglobin (MW_theoretical = 16951) that was: A) frozen, thawed, then incubated at room temperature for 2 h in presence of 500 mM H₂O₂ prior to catalase treatment, B) incubated at −200 C for 2 h after removal of H₂O₂ by catalase, C) thawed, and catalase-treated after incubation with 500 mM H₂O₂ for 2 h at −200 C, D) freeze-dried for 0.5 h to remove the 500 mM H₂O₂, then resuspended in water for MS analysis.

Figure 2

20 μM Apomyoglobin incubated for 2 h at −80 °C in varying concentrations of H₂O₂: A) 0 mM; B) 50 mM H₂O₂; C) 75 mM; D) 100 mM; E) 250 mM; F) 500 mM.

Figure 3

20 μM apomyoglobin incubated for 2 h at −15 °C with varying concentrations of H₂O₂: A) 0 mM; B) 50 mM H₂O₂; C) 75-mM; D) 100 mM; E) 250 mM; F) 500-mM.

Figure 4

Time course of a −80 °C incubation of 20-μM apomyoglobin with 100 mM H₂O₂ A) 30 min; B) 1 h; C) 2 h; D) 3 h; E) 24 h.

Figure 5

Mass spectra after A) a 2 h, −80 °C incubation of 20 μM apomyoglobin with 100 mM H₂O₂; B) same as A) but with phenylalanine added to 10 mM; C) same as A) but with phenylalanine added to 70 mM.

Figure 6

Space-filling model of Val 17 with His 24 and Trp 14 showing no more than 5 Å separation and permitting radical transfer from aromatic H24 or W14 to V17.