Autor: Christian Schöneich, Susan W. Hovorka, Todd D. Williams, Andreas F.R. Hühmer, Homigol Biesiada
Rok vydání: 2002
Předmět:
Zdroj: Pharmaceutical Research. 19:530-537
ISSN: 0724-8741
DOI: 10.1023/a:1015164200431
Popis: 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 5HisB and 10HisB 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 μM INS/8.8μM Zn2+ were exposed to 8 μM Cu2+ and 50 μM 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 10HisB-ox/5HisB-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 10HisB possessing ca. three times greater susceptibility to MCO than 5HisB.
Databáze: OpenAIRE
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