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. 1981 Dec 1;199(3):557–564. doi: 10.1042/bj1990557

N-terminal sequence analysis of polypeptide at the picomole level.

J Y Chang
PMCID: PMC1163410  PMID: 6803770

Abstract

This paper describes a manual method for N-terminal sequence analysis of polypeptides at subnanomole sensitivity. The polypeptide is degraded stepwise by using the dimethylaminoazobenzene isothiocyanate/phenyl isothiocyanate double-coupling method, and the released dimethylaminoazobenzenethiohydantoins of amino acids were identified by reversed-phase high-pressure liquid chromatography. The dimethylaminoazobenzenethiohydantoins are coloured compounds and can be detected in the visible region with the sensitivity limit of 1 pmol (signal-to-baseline noise ratio 5). A high-pressure liquid-chromatographic method was developed for complete analysis of all amino acid dimethylaminoazobenzenethiohydantoin derivatives, including the by-products of serine and threonine. Thus, without use of an automatic sequenator or radioactive materials, it is possible to determine the complete sequence of peptides and N-terminal sequence of proteins with less than 1 nmol of material.

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Selected References

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

  1. Allen G., Fantes K. H. A family of structural genes for human lymphoblastoid (leukocyte-type) interferon. Nature. 1980 Oct 2;287(5781):408–411. doi: 10.1038/287408a0. [DOI] [PubMed] [Google Scholar]
  2. Allen G., Wittmann-Liebold B. The amino acid sequence of the ribosomal protein S8 of Escherichia coli. Hoppe Seylers Z Physiol Chem. 1978 Nov;359(11):1509–1525. doi: 10.1515/bchm2.1978.359.2.1509. [DOI] [PubMed] [Google Scholar]
  3. Bridgen J. High sensitivity amino acid sequence determination. Application to proteins eluted from polyacrylamide gels. Biochemistry. 1976 Aug 10;15(16):3600–3604. doi: 10.1021/bi00661a030. [DOI] [PubMed] [Google Scholar]
  4. Chang J. Y. Amino-terminal analysis of polypeptide using dimethylaminoazobenzene isothiocyanate. Anal Biochem. 1980 Mar 1;102(2):384–392. doi: 10.1016/0003-2697(80)90172-4. [DOI] [PubMed] [Google Scholar]
  5. Chang J. Y. High-sensitivity sequence analysis of peptides and proteins by 4-NN-dimethylaminoazobenzene 4'-isothiocyanate. Biochem J. 1977 Jun 1;163(3):517–520. doi: 10.1042/bj1630517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chang J. Y. Isolation and characterization of polypeptide at the picomole level. Pre-column formation of peptide derivatives with dimethylaminoazobenzene isothiocyanate. Biochem J. 1981 Dec 1;199(3):537–545. doi: 10.1042/bj1990537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chang J. Y., Knecht R., Braun D. G. Amino acid analysis at the picomole level. Application to the C-terminal sequence analysis of polypeptides. Biochem J. 1981 Dec 1;199(3):547–555. doi: 10.1042/bj1990547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chang J. Y. Manual solid phase sequence analysis of polypeptides using 4-N-N,-dimethylaminoazobenzene 4'-isothiocyanate. Biochim Biophys Acta. 1979 May 23;578(1):188–195. doi: 10.1016/0005-2795(79)90126-0. [DOI] [PubMed] [Google Scholar]
  9. Chang J. Y. The destruction of serine and threonine thiohydantoins during the sequence determination of peptides by 4-N,N-dimethylaminoazobenzene 4'-isothiocyanate. Biochim Biophys Acta. 1979 May 23;578(1):175–187. doi: 10.1016/0005-2795(79)90125-9. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Hughes G. J., Winterhalter K. H., Lutz H., Wilson K. J. Microsequence analysis: III. Automatic solid-phage sequencing using DABITC. FEBS Lett. 1979 Dec 1;108(1):92–97. doi: 10.1016/0014-5793(79)81185-0. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Hunkapiller M. W., Hood L. E. New protein sequenator with increased sensitivity. Science. 1980 Feb 1;207(4430):523–525. doi: 10.1126/science.7352258. [DOI] [PubMed] [Google Scholar]
  14. Klapper D. G., Wilde C. E., 3rd, Capra J. D. Automated amino acid sequence of small peptides utilizing Polybrene. Anal Biochem. 1978 Mar;85(1):126–131. doi: 10.1016/0003-2697(78)90282-8. [DOI] [PubMed] [Google Scholar]
  15. Laursen R. A. Solid-phase Edman degradation. An automatic peptide sequencer. Eur J Biochem. 1971 May 11;20(1):89–102. doi: 10.1111/j.1432-1033.1971.tb01366.x. [DOI] [PubMed] [Google Scholar]
  16. Lewis U. J., Bonewald L. F., Lewis L. J. The 20,000-dalton variant of human growth hormone: location of the amino acid deletions. Biochem Biophys Res Commun. 1980 Jan 29;92(2):511–516. doi: 10.1016/0006-291x(80)90363-0. [DOI] [PubMed] [Google Scholar]
  17. Richardson M. The complete amino acid sequence and the trypsin reactive (inhibitory) site of the major proteinase inhibitor from the fruits of aubergine (Solanum melongena L.). FEBS Lett. 1979 Aug 15;104(2):322–326. doi: 10.1016/0014-5793(79)80843-1. [DOI] [PubMed] [Google Scholar]
  18. Tarr G. E., Beecher J. F., Bell M., McKean D. J. Polyquarternary amines prevent peptide loss from sequenators. Anal Biochem. 1978 Feb;84(2):622–7?0=ENG. doi: 10.1016/0003-2697(78)90086-6. [DOI] [PubMed] [Google Scholar]
  19. Wittmann-Liebold B. Amino acid sequence studies on ten ribosomal proteins of Escherichia coli with an improved sequenator equipped with an automatic conversion device. Hoppe Seylers Z Physiol Chem. 1973 Oct-Nov;354(10-11):1415–1431. doi: 10.1515/bchm2.1973.354.2.1415. [DOI] [PubMed] [Google Scholar]
  20. Wittmann-Liebold B. Primary structure of protein L24 from the Escherichia coli ribosome. FEBS Lett. 1979 Dec 1;108(1):75–80. doi: 10.1016/0014-5793(79)81182-5. [DOI] [PubMed] [Google Scholar]
  21. Zimmerman C. L., Appella E., Pisano J. J. Rapid analysis of amino acid phenylthiohydantoins by high-performance liquid chromatography. Anal Biochem. 1977 Feb;77(2):569–573. doi: 10.1016/0003-2697(77)90276-7. [DOI] [PubMed] [Google Scholar]

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