Purpose: To establish the sensitivity of Zn2+ insulin (Zn2+-INS) to metal-catalyzed oxidation (MCO) and to use tandem mass spectrometry/mass spectroscopy (MS/MS) for the identification and quantification of 2-oxo-histidine at 5His, and 10His(B) upon the MCO of INS.
Methods: Zn2+-INS was exposed to Cu2+/ascorbate-induced MCO. Products were analyzed by reverse-phase high-performance liquid chromatography, electrospray ionization mass spectrometry (ESI-MS) and MS/MS, sodium dodecylsulfate polyacrylamide gel electrophotesis (SDS-PAGE), and CD spectroscopy.
Results: A maximal loss of 40% INS was achieved when 20 microM INS/8.81 microM Zn2+ were exposed to 8 microM Cu2+ and 50 microM ascorbate. MCO was completely inhibited by ethylenediaminetetraacetic acid or native catalase but not with a 1000-fold molar excess of Zn2+ over Cu2+. MCO did not alter the aggregation state of INS. High-performance liquid chromatography-fractionated products contained portions of oxidized and native INS monomers. Oxidation selectively targeted the B chain of INS, where MS/MS sequencing revealed 2-oxo-His formation at both His residues at a relative ratio of 10His(B-ox/5His(B-ox) = 2.8 +/- 1.3 (SD).
Conclusions: At a Zn2+/INS molar ratio comparable to that in regular INS preparations, Zn2+-INS was susceptible to MCO. Both His residues of INS were converted partially to 2-oxo-His, with 10His(B) possessing ca. three times greater susceptibility to MCO than 5His(B).