Abstract

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).

Highlights

  • The succinimide variant was isolated from thermally stressed recombinantmethionyl human growth hormone(hGH)by high performance anionexchange chromatography, further purified by reversed-phase high performance liquid chromatogra

  • A difference of 18 u was 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)

  • Johnson et al [9] used protein carboxyl methyltransferase, an enzyme that methylates isoaspartyl linkages, to determine which sites in Met-hGH are prone to deamidation/isomerization

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Summary

DISCUSSION

Deamidation a t Asn-149 and isomerization at Asp-130 have been shown to be the major sites of deamidation/isomerization in hGH [9]. Our results demonstrate thata succinimide is an intermediate in the isomerization of a-aspartate to @aspartate at position 130. The difference in charge between the native protein and the succinimide variant allowed their resolution by ion-exchange chromatography. The producotsf deamidation at Asn-149 (01- and @-aspartate)have been previously identified; thepresence of asuccinimide intermediate at this position has nbeoetn demonstrated. This is not unexpected since, in this case, thesuccinimide variant and native proteinhave the samecharge. It was fortuitous, in this study, thatAsp-130 of hGH is vulnerable to isomerization. In order to isolate sufficient quantities of the more basic component for characterization studies, 1 mg of thermally degraded Met-hGH was loaded onto a DEAE-3SW column

SucciFniomrmidaetion in HGHurmorwamnthone
TRYPTIC MAPPING
RESULTS
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