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The oxidation of recombinant human interleukin-2 (rhlL-2) by potassium peroxodisulfate (KPS) with or without N,N,N',N'-tetramethylethylenediamine (TEMED), which are used for the preparation of dextran-based hydrogels, was investigated.The oxidation of (derivatives of) methionine. tryptophan, histidine and tyrosine, as well as rhlL-2 was investigated. Both the oxidation kinetics (RP-HPLC) and the nature of the oxidation products (mass spectrometry) were studied as a function of the KPS and TEMED concentration, and the presence of a competitive antioxidant, methionine.Under conditions relevant for the preparation of rhIL-2 loaded hydrogels, only methionine and tryptophan derivatives were susceptible to oxidation by KPS. The oxidation of these compounds was inhibited once TEMED was present, suggesting that the peroxodisulfate anion, rather than the radicals formed in the presence of TEMED, is the oxidative species. KPS only induced oxidation of the four methionines present in rhIL-2, whereas the tryptophan residue remained unaffected. The radicals, formed after KPS decomposition by TEMED, induced some dimerization of rhIL-2. The oxidation of rhIL-2 could be substantially reduced by the addition of methionine, or by pre-incubation of KPS with TEMED.Only the methionine residues in rhlL-2 are oxidized by KPS. The extent of oxidation can be minimized by a proper selection of the reaction conditions. |
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