J. Biol. Chem., Vol. 266, Issue 21, 13544-13547, 07, 1991
Isolation and characterization of a succinimide variant of methionyl human growth hormone
G Teshima, JT Stults, V Ling and E Canova-Davis
Department of Medicinal and Analytical Chemistry, , Genentech, Inc., South San Francisco, California 94080.
Deamidation of asparagine and glutamine residues, isomerization of aspartic
acid side chains, and racemization of the L- to the D-form of the amino
acids are common spontaneous chemical reactions known to occur in proteins.
Previous studies have implicated succinimides as intermediates in these
reactions; however, the evidence has been indirect. Our results
demonstrate, for the first time, the presence of a succinimide intermediate
in an intact protein. The succinimide (cyclic imide) variant was isolated
from thermally stressed recombinant methionyl human growth hormone (hGH) by
high performance anion-exchange chromatography, further purified by
reversed-phase high performance liquid chromatography, and analyzed by
tryptic mapping. A later eluting tryptic peptide, compared with the native
T12 peptide (residues 128- 134, Leu-Glu-Asp-Gly-Ser-Pro-Arg), was analyzed
by mass spectrometry (MS). This variant had a protonated molecular mass of
755.3 atomic mass units (u), as compared with 773.3 u for the native T12
peptide. A difference of 18 u, a loss of water, is consistent with the
formation of a succinimide intermediate at Asp-130 of methionyl hGH. MS/MS
analysis of the cyclic imide-containing peptide verified that the
modification occurred at Asp-130. A difference of 18 u was also observed
for the intact cyclic imide methionyl hGH variant (22,238 u), as measured
by electrospray mass spectrometry, compared with native methionyl hGH
(22,256 u).
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