Chemical Modifications in Aggregates of Recombinant Human Insulin Induced by Metal-Catalyzed Oxidation: Covalent Cross-Linking via Michael Addition to Tyrosine Oxidation Products

Abstract

PurposeTo elucidate the chemical modifications in covalent aggregates of recombinant human insulin induced by metal catalyzed oxidation (MCO).MethodsInsulin was exposed for 3 h at room temperature to the oxidative system copper(II)/ascorbate. Chemical derivatization with 4-(aminomethyl) benzenesulfonic acid (ABS) was performed to detect 3,4-dihydroxyphenylalanine (DOPA) formation. Electrospray ionization-mass spectrometry (ESI-MS) was employed to localize the amino acids targeted by oxidation and the cross-links involved in insulin aggregation. Oxidation at different pH and temperature was monitored with size exclusion chromatography (SEC) and ESI-MS analysis to further investigate the chemical mechanism(s), to estimate the aggregates content and to quantify DOPA in aggregated insulin.ResultsThe results implicate the formation of DOPA and 2-amino-3-(3,4-dioxocyclohexa-1,5-dien-1-yl) propanoic acid (DOCH), followed by Michael addition, as responsible for new cross-links resulting in covalent aggregation of insulin during MCO. Michael addition products were detected between DOCH at positions B16, B26, A14, and A19, and free amino groups of the N-terminal amino acids Phe B1 and Gly A1, and side chains of Lys B29, His B5 and His B10. Fragments originating from peptide bond hydrolysis were also detected.ConclusionMCO of insulin leads to covalent aggregation through cross-linking via Michael addition to tyrosine oxidation products.Electronic supplementary materialThe online version of this article (doi:10.1007/s11095-012-0755-z) contains supplementary material, which is available to authorized users.

Identification of Oxidation Sites and Covalent Cross-Links in Metal Catalyzed Oxidized Interferon Beta-1a: Potential Implications for Protein Aggregation and Immunogenicity

“…Results of the current study, in particular, reveal that the oxidative modification of Phe and Tyr results in an electron acceptor structure, namely DOCH, which may be involved in IFNβ1a cross-linking through a 1,4- or 1,6-type addition mechanism of primary amines to DOCH. The results reported in this work are in agreement with the data we recently published using insulin as a model protein (17) . However, since Met and Trp are not present in insulin, we did not know whether these are involved in covalent cross-linking of other proteins such as IFNβ1a.…”

“…SEC with fluorescence detection of ABS-tagged proteins was used to study whether the monomers or aggregates contain DOPA (3,4-dihydroxyphenylalanine) and/or DOCH (2-amino-3-(3,4-dioxocyclohexa-1,5-dien-1-yl) propanoic acid), produced by oxidation of tyrosine (Tyr) and/or phenylalanine (Phe) residues and subject to fluorogenic derivatization with ABS (17) and/or 5-hydroxytryptophan, a Trp oxidation product which can also form fluorescent benzoxazole products upon ABS derivatization (27, 28) . Our data show that after derivatization of the oxidized and dialyzed protein with ABS, the total SEC fluorescence peak area (i.e.…”

“…To obtain oxidized IFNβ1a, untreated IFNβ1a, diluted to 200 µg/ml with PBS, was incubated with 4 mM ascorbic acid and 40 μM CuCl 2 for 3 hours at room temperature. The oxidation reaction was stopped by adding 100 mM EDTA to a final concentration of 1 mM as previously reported (17) .…”

Mouse Models for Assessing Protein Immunogenicity: Lessons and Challenges

“…23,25,40,60,62 Moreover, all of these aggregates showed some oxidation of histidine 61,63 and the aromatic amino acid residues. 63,64 Hydrogen peroxide-mediated oxidation, 21,40 stirring 61 and UV-light exposure 22,24,28 have also been used to generate protein aggregates which have enhanced immunogenicity in the murine model system tested compared to that of the protein before stress treatment. It is important to note that all of the chemical modifications described above were induced by treatments that increased the amount of such modifications to levels that and are not representative of what typically would be seen in the clinic.…”

Metal-Catalyzed Oxidation of Protein Methionine Residues in Human Parathyroid Hormone (1-34): Formation of Homocysteine and a Novel Methionine-Dependent Hydrolysis Reaction

“…Indeed, the oxidation of Tyr and Phe residues can mediate the formation of cross-links with amino groups through Michael addition. 17 …”