Reversed phase HPLC of Met58 oxidized rhIL-11: oxidation enhanced by plastic tubes.

Abstract

The hydrophobicity of human recombinant interleukin 11 (rhIL-11) with an oxidized Met58 residue is nearly identical to the hydrophobicity of native rhIL-11. Consequently, separation of these species using standard gradient elution or isocratic elution is very difficult. Using an optimized, shallow gradient RP-HPLC method. Met58 oxidized rhIL-11 could be separated sufficiently from native rhIL-11. The identity of the oxidized form detected with this method was confirmed by peptide mapping with trypsin and endoproteinase Asp-N, N-terminal sequencing and mass spectrometric analysis. This method was employed to determine the effect of disposable laboratory plastic tubes for the oxidation. The amounts of Met58 oxidized rhIL-11 were increased when rhIL-11 samples were stored in plastic tubes at 37 degrees C in the dark. Samples stored in polypropylene tubes were oxidized much more than samples stored in polystyrene tubes. Additionally, the oxidation was greatly enhanced when samples were stored in polypropylene tubes exposed to light before rhIL-11 sample storage. The extent of the oxidation was also affected by the sources of polypropylene tubes. A maximum increase in Met58 oxidized rhIL-11 was more than 30% when samples were stored at 37 degrees C for 14 days in polypropylene tubes exposed to a daylight fluorescent lamp for 25 days. Consequently, these results indicate that attention should be paid for selection of suitable plastic tubes used for storage of protein samples, and for protection of the plastic tubes themselves from extended exposure to light while in storage.