Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1981 Dec 1;199(3):547–555. doi: 10.1042/bj1990547

Amino acid analysis at the picomole level. Application to the C-terminal sequence analysis of polypeptides.

J Y Chang, R Knecht, D G Braun
PMCID: PMC1163409  PMID: 6803769

Abstract

Amino acids labelled with dimethylaminoazobenzenesulphonyl chloride can be separated by reversed-phase high-pressure liquid chromatography and detected in the visible region (436 nm). All 19 naturally occurring amino acids can be separated on a Zorbax ODS column by employing two different gradient systems consisting of an acetonitrile/aqueous buffer mixture. As little as 2--5 pmol of an individual dimethylaminoazobenzenesulphonyl-amino acid can be quantitatively analysed with reliability, and only 10--30 ng of the dimethylaminoazobenzenesulphonylated protein hydrolysate is needed for each complete amino acid analysis. This new technique is as sensitive as any of the current amino acid analysis methods involving ion-exchange separation plus fluorescence detection, and is technically much simpler. By the combination of this sensitive amino acid-analysing technique with carboxypeptidase, we have been able to determine the C-terminal sequence of polypeptides at the picomole level.

Full text

PDF
555

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Benson J. R., Hare P. E. O-phthalaldehyde: fluorogenic detection of primary amines in the picomole range. Comparison with fluorescamine and ninhydrin. Proc Natl Acad Sci U S A. 1975 Feb;72(2):619–622. doi: 10.1073/pnas.72.2.619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Edman P., Begg G. A protein sequenator. Eur J Biochem. 1967 Mar;1(1):80–91. doi: 10.1007/978-3-662-25813-2_14. [DOI] [PubMed] [Google Scholar]
  3. Hare P. E. Subnanomole-range amino acid analysis. Methods Enzymol. 1977;47:3–18. doi: 10.1016/0076-6879(77)47003-4. [DOI] [PubMed] [Google Scholar]
  4. Hayashi R. Carboxypeptidase Y in sequence determination of peptides. Methods Enzymol. 1977;47:84–93. doi: 10.1016/0076-6879(77)47010-1. [DOI] [PubMed] [Google Scholar]
  5. Hill D. W., Walters F. H., Wilson T. D., Stuart J. D. High performance liquid chromatographic determination of amino acids in the picomole range. Anal Chem. 1979 Jul;51(8):1338–1341. doi: 10.1021/ac50044a055. [DOI] [PubMed] [Google Scholar]
  6. Hunkapiller M. W., Hood L. E. Direct microsequence analysis of polypeptides using an improved sequenator, a nonprotein carrier (polybrene), and high pressure liquid chromatography. Biochemistry. 1978 May 30;17(11):2124–2133. doi: 10.1021/bi00604a016. [DOI] [PubMed] [Google Scholar]
  7. Lee H. M., Forde M. D., Lee M. C., Bucher D. J. Fluorometric microbore amino acid analyzer: the construction of an inexpensive, highly sensitive instrument using o-phthalaldehyde as a detection agent. Anal Biochem. 1979 Jul 15;96(2):298–307. doi: 10.1016/0003-2697(79)90585-2. [DOI] [PubMed] [Google Scholar]
  8. Lee K. S., Drescher D. G. Derivatization of cysteine and cystine for fluorescence amino acid analysis with the o-phthaldialdehyde/2-mercaptoethanol reagent. J Biol Chem. 1979 Jul 25;254(14):6248–6251. [PubMed] [Google Scholar]
  9. Lin J. K., Chang J. Y. Chromophoric labeling of amino acids with 4-dimethylaminoazobenzene-4'-sulfonyl chloride. Anal Chem. 1975 Aug;47(9):1634–1638. doi: 10.1021/ac60359a007. [DOI] [PubMed] [Google Scholar]
  10. MOORE S., STEIN W. H. Procedures for the chromatographic determination of amino acids on four per cent cross-linked sulfonated polystyrene resins. J Biol Chem. 1954 Dec;211(2):893–906. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES