High sensitivity of Zn2+ insulin to metal-catalyzed oxidation: detection of 2-oxo-histidine by tandem mass spectrometry.
OBJECTIVE
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).
Hovorka
Susan W
SW
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence 66047, USA.
Biesiada
Homigol
H
Williams
Todd D
TD
Hühmer
Andreas
A
Schöneich
Christian
C
D002384:Catalysis / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid / Q000379:methods / Q000706:statistics & numerical data; D006639:Histidine / Q000031:analogs & derivatives* / Q000032:analysis* / Q000737:chemistry; D006801:Humans; D007328:Insulin / Q000032:analysis / Q000737:chemistry*; D019216:Metals, Heavy / Q000494:pharmacology*; D010084:Oxidation-Reduction / Q000187:drug effects; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D015032:Zinc / Q000032:analysis / Q000737:chemistry*
D007328:Insulin; D019216:Metals, Heavy; D011994:Recombinant Proteins; C083725:2-oxohistidine; D006639:Histidine; D015032:Zinc