Micro-Isolation of Polypeptides Precolumn Labeled with Hydrophobic Chromophore

  • Chang Jui-Yoa

Abstract

Detection of polypeptides is a crucial technique associated with every Chromatographie methods of peptide isolation. Conventionally, peptides are detected, after Chromatographie separation, by their reaction with reagents like ninhydrin [1, 2], fluorescamine [3, 4] or o-phthaldialdehyde [5, 6]. Alternatively, peptides can be detected through their intrinsic absorption of amide bonds at low UV region (200 nm–230 nm) [7,8]. This low UV detection system in combination with the use of reversed phase HPLC is at present the most versatile technique for the isolation of peptides. To enhance the sensitivity of detection, peptides can also be labeled with chromophore preceding Chromatographie separation. For instance, dansylated peptide mappings [9, 10]. We have developed two precolumn labeling techniques for micro-isolation of peptides. (1) A method using DABITC labeling for isolation of peptides with free N-termini [11]. (2) A method using DABIA labeling for selective isolation of cysteine containing peptides [12].

Keywords

HPLC Acetonitrile Cysteine Pyridine Polypeptide 

Abbreviations

DABITC

dimethylaminoazobenzene isothiocyanate

DABTC

dimethylamino-azobenzene thiocarbamoyl

DABTH

dimethylaminoazobenzene thiohydantoin

DABIA

dimethylaminoazobenzene iodoacetamide

DABCAM

dimethylaminoazobenzene carboxyamidomethyl

PITC

phenylisothiocyanate

PTH

phenylthiohydantoin

TFA

trifluoroacetic acid

HPLC

high performance liquid chromatography

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References

  1. 1.
    Heinz N, Hultin T (1983) Methods Enzymol 91:359–366.CrossRefGoogle Scholar
  2. 2.
    Hermann AC, Vanaman TC (1977) Methods Enzymol 47:220–236.CrossRefGoogle Scholar
  3. 3.
    Lai CY (1977) Methods Enzymol 37:236–243.CrossRefGoogle Scholar
  4. 4.
    Samejima K, Dairman W, Udenfriend S (1971) Anal Bioehem 42:222–229.CrossRefGoogle Scholar
  5. 5.
    Roth M (1971) Anal Chem 43:880–884.PubMedCrossRefGoogle Scholar
  6. 6.
    Bensen JR, Hare PE (1975) Proc Natl Acad Sci USA 72:619–622.CrossRefGoogle Scholar
  7. 7.
    Machleidt W, Otto J, Wächter E (1977) Methods Enzymol 47:210–220.PubMedCrossRefGoogle Scholar
  8. 8.
    Hermodson M, Mahoney WC (1983) Methods Enzymol 91:352–359.PubMedCrossRefGoogle Scholar
  9. 9.
    Tichy H (1975) Anal Biochem 69:552–557.PubMedCrossRefGoogle Scholar
  10. 10.
    Zanetta JP (1970) J Chromatogr 51:441–458.PubMedCrossRefGoogle Scholar
  11. 11.
    Chang J-Y (1981) Biochem J 199:537–545.PubMedGoogle Scholar
  12. 12.
    Chang J-Y, Knecht R, Braun DG (1983) Biochem J 211:163–171.PubMedGoogle Scholar
  13. 13.
    Chang J-Y, Herbst H, Aebersold R, Braun DG (1983) Biochem J 211:173–180.PubMedGoogle Scholar
  14. 14.
    Chang J-Y, Knecht R, Ball R, Alkan SS, Braun DG (1982) Eur J Biochem 127:625–629.PubMedCrossRefGoogle Scholar
  15. 15.
    Chang J-Y, Knecht R, Maschler R, Seemuller U (1985) Biol Chem Hoppe-Seyler 366:281–286.PubMedCrossRefGoogle Scholar
  16. 16.
    Chang J-Y, Tran TH (1986) J Biol Chem 261:1174–1176.PubMedGoogle Scholar
  17. 17.
    Edman P, Begg G (1967) Eur J Biochem 1:80–91.PubMedCrossRefGoogle Scholar
  18. 18.
    Chang J-Y (1983) Methods Enzymol 91:455–466.PubMedCrossRefGoogle Scholar
  19. 19.
    Chang J-Y, Brauer D, Wittmann-Liebold B (1978) FEBS Lett 93:205–214.CrossRefGoogle Scholar
  20. 20.
    Chang J-Y (1978) FEBS Lett 91:63–68.PubMedCrossRefGoogle Scholar
  21. 21.
    Dodt J, Müller H-P, Seemuller U, Chang J-Y (1984) FEBS Lett 165:180–184.CrossRefGoogle Scholar
  22. 22.
    Gurd FRN (1972) Methods Enzymol 25:424–438.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg. 1986

Authors and Affiliations

  • Chang Jui-Yoa
    • 1
  1. 1.Pharmaceutical Research LaboratoriesCiba-Geigy LimitedBaselSwitzerland

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