Isolation and characterization of a succinimide variant of methionyl human growth hormone
| Autor: | Eleanor Canova-Davis, Glen Teshima, Victor Ling, John T. Stults |
|---|---|
| Rok vydání: | 1991 |
| Předmět: |
Hot Temperature
Molecular Sequence Data Succinimides Peptide In Vitro Techniques Biochemistry Mass Spectrometry chemistry.chemical_compound Methionine Succinimide Aspartic acid Humans Trypsin Amino Acid Sequence Imide Deamidation Molecular Biology Peptide sequence Chromatography High Pressure Liquid chemistry.chemical_classification Aspartic Acid Chromatography Molecular mass Cell Biology Peptide Fragments Amino acid chemistry Growth Hormone |
| Zdroj: | Journal of Biological Chemistry. 266:13544-13547 |
| ISSN: | 0021-9258 |
| Popis: | 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). |
| Databáze: | OpenAIRE |
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