Synthesis of stereoisomers and isoforms of a tryptic heptapeptide fragment of human growth hormone and analysis by reverse-phase HPLC and capillary electrophoresis.
The amino acid sequence Asn-Gly has at pH 7 a tendency to induce deamidation of asparagine to aspartic acid via the formation of a cyclic imide. This imide opens up to yield Asp-Gly or the isoaspartic acid (isoAsp) form, isoAsp-Gly. Both isomers may be found in their L-form or D-form. Like Asn-Gly, the sequence Asp-Gly has a tendency for isomerization and racemization via the formation of a cyclic imide intermediate. When human growth hormone is digested with trypsin, one of the fragments is a heptapeptide (amino acid residues 128-134) containing the amino acid sequence Asp-Gly (amino acid residues 130 and 131). This heptapeptide, as well as stereoisomers and isoforms where L-Asp was replaced by D-Asp, L-isoAsp, D-isoAsp or the L-cyclic imide, respectively, has been synthesized and used as a standard to achieve separation of the five forms by capillary electrophoresis and by reverse-phase HPLC. Capillary electrophoresis analysis was performed in uncoated capillaries by the use of aspartic acid/cyclodextrin buffers at low pH. The elution order of the aspartic-acid-containing heptapeptides was D-Asp, L-Asp, L-isoAsp, D-isoAsp and L-cyclic imide. Reverse-phase HPLC analysis was performed on a C18 column by the use of a shallow acetonitrile gradient in trifluoroacetic acid/water. The elution order was D-isoasp, L-isoASp, L-Asp, D-Asp and L-cyclic imide. Human growth hormone samples were degraded by incubation at high temperature and analyzed for their potential content of isomerization and racemization products. Only L-forms of aspartic acid and isoaspartic acid of the heptapeptide fragment were found.
Vinther
A
A
Novo Nordisk A/S, Quality Control Laboratories, Niels Steensensvej, Gentofte, Denmark.
Holm
A
A
Høeg-Jensen
T
T
Jespersen
A M
AM
Klausen
N K
NK
Christensen
T
T
Sørensen
H H
HH
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D019075:Electrophoresis, Capillary; D013006:Growth Hormone / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics; D013237:Stereoisomerism; D014357:Trypsin
D009842:Oligopeptides; D010446:Peptide Fragments; D013006:Growth Hormone; D014357:Trypsin