Micro-Determination of Amino Acids and Related Compounds with Dansyl Chloride

  • Volker Neuhoff
Part of the Molecular Biology Biochemistry and Biophysics book series (MOLECULAR, volume 14)


Dansyl chloride (Dans-Cl, l-dimethylamino-naphthalene-5-sulfonyl chloride) was used by Weber in 1952 for the first time for the preparation of fluorescent conjugates of albumin. It has subsequently found almost as wide an application as Fischer’s naphthalene sulfonyl-chloride or Sanger’s 2,4-dinitroflurobenzene (reviews, see Gray, 1967a; Seiler, 1970; Seiler and Wiechmann, 1970). Its usefulness is due to the fact that its reaction products with amino acids, amines, peptides, proteins, phenols, imidazoles, and sulphydryl groups have an intense yellow to yellow-orange fluorescence and can be separated easily with suitable chromatographic systems (Seiler, 1970). Woods and Wang (1967) first described the fractionation of dansylated amino acids on polyamide sheets (review see Wang and Weinstein, 1972), and Gray and Hartley (1969) introduced this method of separation for the determination of end-groups and in sequence analysis of proteins and peptides. It was Hartley who, during an EMBO summer- school in Elmau in 1969, suggested adapting this technique to the micro-scale. This was immediately successful, the normal 15 × 15 cm polyamide sheets were simply replaced by 3 × 3 cm ones, and the application of the dansylated sample was performed with a very fine capillary under the stereomicroscope. On a 15 × 15 cm polyamide layer about 10-9 moles of each dansylated amino acid is detectable; using 3 × 3 cm polyamide layers as little as 10-12 moles can be detected.


Excitation Spectrum Free Amino Acid Individual Amino Acid Kynurenic Acid Helix Pomatia 
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  1. Ambler, R.P.: Enzymic hydrolysis with carboxypeptidases. In: Methods in enzymology, vol. XI, Enzyme structure, ed. by C. H.W. Hirs, p. 155–166. New York-London: Academic Press 1967.Google Scholar
  2. Bell, C.E., Somerville, A. R.: A new fluorescence method for detection and possible quantitative assay of some catecholamine and tryptamine derivatives on paper. Biochem. J. 98, 1c–3c (1966).PubMedGoogle Scholar
  3. Braunitzer, G.: Bestimmung der Reihenfolge der Aminosäuren am Carboxylende des Tabakmosaikvirus durch Hydrazinspaltung. Chem. Ber. 88, 2025–2036 (1955).CrossRefGoogle Scholar
  4. Briel, G.: Mikroanalyse von Aminosäuren als 14C-Dansyl-Verbindungen und ihre Anwendung zur Bestimmung von freien Aminosäuren des Zentralnervensystems. Inaugural-Dissertation, Göttingen 1972.Google Scholar
  5. Briel, G., Gylfe, E., Hellmann, B., Neuhoff, V.: Microdetermination of free amino acids in pancreatic islets isolated from obese-hyperglycemic mice. Acta physiol. scand. 84, 247–253 (1972).PubMedCrossRefGoogle Scholar
  6. Briel, G., Neuhoff, V.: Microanalysis of amino acids and their determination in biological material using dansyl chloride. Hoppe-Seylers Z. physiol. Chem. 353, 540–553 (1972).PubMedCrossRefGoogle Scholar
  7. Briel, G., Neuhoff, V., Osborne, N.N.: Determination of amino acids in single identifiable nerve cells of Helix pomatia. Int. J. Neurosci. 2, 129–136 (1971).PubMedCrossRefGoogle Scholar
  8. Bruton, C.J., Hartley, B.S.: Chemical studies on methionyl-tRNA synthetase from Escherichia coli. J. molec. Biol. 52, 165–178 (1970).PubMedCrossRefGoogle Scholar
  9. Burgess, R. R.: Separation and characterization of the subunits of ribonucleic acid polymerase. J. biol. Chem. 244, 6168–6178 (1969).PubMedGoogle Scholar
  10. Crowshaw, K., Jessup, J., Ramwell, P. W.: Thin-layer chromatography of 1-dimethylamino- naphthalene-5-sulphonyl derivatives of amino acids present in superfusates of cat cerebral cortex. Biochem. J. 103, 79–85 (1967).PubMedGoogle Scholar
  11. Curtis, D. R.: Central synaptic transmitters. Proc. Aust. Assoc. Neurologists 7, 55–60 (1970).Google Scholar
  12. Davidson, A.N., Kacmarek, L.K.: Taurine—a possible neurotransmitter? Nature (Lond.) New Biol. 234, 107–108 (1971).CrossRefGoogle Scholar
  13. Fraenkel-Conrat, H., Chun Ming Tsung: Hydrazinolysis, In: Methods in enzymology, vol. XI, Enzyme structure, ed. by C.H.W. Hirs, p. 151–155. New York-London: Academic Press 1967.Google Scholar
  14. Frey, W.: Freie Aminosäuren der Sehbahn des Kaninchens. Inaugural-Dissertation, Göttingen 1972.Google Scholar
  15. Gray, W. R.: Ultra-micro methods for investigation of protein structure. Ph. D. Thesis, St. John’s College, University of Cambridge 1964.Google Scholar
  16. Gray, W. R.: Dansyl chloride procedure, In: Methods in enzymology, vol. XI, Enzyme structure, ed. by C.H.W. Hirs, p. 139–151. New York-London: Academic Press 1967a.Google Scholar
  17. Gray, W. R.: Sequential degradation plus dansylation. In: Methods in enzymology, vol. XI, Enzyme structure, ed. by C.H.W. Hirs, p.469–475. New York-London: Academic Press 1967b.Google Scholar
  18. Gray, W. R., Hartley, B.S.: A fluorescent end-group reagent for proteins and peptides. Biochem. J. 89, 59p (1963).Google Scholar
  19. Gros, C., Labouesse, B.: Study of the dansylation reaction of amino acids, peptides and proteins. Europ. J. Biochem. 7, 463–470 (1969).PubMedCrossRefGoogle Scholar
  20. Hartley, B.S.: Strategy and tactics in protein chemistry. Biochem. J. 119, 805–822 (1970).PubMedGoogle Scholar
  21. Hartley, B.S., Massey, V.: The active centre of Chymotrypsin. I. Labelling with a fluorescent dye. Biochim. biophys. Acta (Amst.) 21, 58–70 (1956).CrossRefGoogle Scholar
  22. Heller, G.: Über die Hydrolyse der Aminosäure-Ester von Transfer-Ribonucleinsäuren. Dissertation, Göttingen 1966.Google Scholar
  23. Hettler, H.: Charakterisierung von primären und sekundären Aminen mit Pseudosaccharinchlorid. Fresenius Z. anal. Chem. 220, 9–15 (1966).CrossRefGoogle Scholar
  24. Hettler, H.: Chapman-Mumm rearrangement of pseudosaccharinesters. Tetrahedron Letters 15, 1793–1796 (1968).CrossRefGoogle Scholar
  25. Hirs, C.H.W.: The oxydation of ribonuclease with performic acid. J. biol. Chem. 219, 611–621 (1956).PubMedGoogle Scholar
  26. Knox, W.E.: Phenylketonuria. In: The metabolic basis of inherited disease, ed. by Stanburg, J.B., Wyngaarden, J.B., Frederickson, D.S., 2. ed., part 3, chap. 11, p. 266-295. New York-Toronto- Sidney-London 1966.Google Scholar
  27. Matthaei, J.H., Heller, G., Voigt, H.-P., Neth, R., Schöch, G., Kübler, H.: Analysis of the genetic code by amino acid adapting. Genetic elements, ed. by D. Shugar, p. 233–250. New York-London: Academic Press 1967.Google Scholar
  28. Neadle, D.J., Pollit, R.J.: The formation of l-dimethylaminonaphthalene-5-sulphonamide during the preparation of l-dimethylaminonaphthalene-5-sulphonyl-amino acids. Biochem. J. 97, 607–608 (1965).PubMedGoogle Scholar
  29. Neuhoff, V.: First Harden Conf. on the Structure and Biological Role of Proteins. Wye College, 1969.Google Scholar
  30. Neuhoff, V.: Micromethods for protein and enzyme analysis. In: International Symposium VI, Chromatographie Electrophorese, p. 57–62. Bruxelles: Press Académiques Européennes S.C. 1971.Google Scholar
  31. Neuhoff, V., Briel, G., Maelicke, A.: Characterization and micro-determination of histidine as its dansyl compounds. Arzneimittel-Forsch. 21, 104–107 (1971).Google Scholar
  32. Neuhoff, V., Haar, F. von der, Schlimme, E., Weise, M.: Zweidimensionale Chromatographie von Dansyl-aminosäuren im pico-Mol-Bereich, angewandt zu direkten Charakterisierung von Transfer- Ribonucleinsäuren. Hoppe-Seylers Z. physiol. Chem. 350, 121–128 (1969).PubMedCrossRefGoogle Scholar
  33. Neuhoff, V., Kiehl, F.: Dialysiergeräte für Volumen zwischen 10 und 500 μl. Arzneimittel-Forsch. (Drug Res.) 19, 1898–1899 (1969).Google Scholar
  34. Neuhoff, V., Weise, M.: Determination of pico-mole quantities of γ-amino-butyric acid (GABA) and serotonin. Arzneimittel-Forsch. (Drug Res.) 20, 368–372 (1970).Google Scholar
  35. Neuhoff, V., Weise, M., Sternbach, H.: Micro-analysis of pure deoxyribonucleic acid-dependent ribonucleic acid polymerase from Escherichia coli. VI. Determination of the amino acid composition. Hoppe Seylers Z. physiol. Chem. 351, 1395–1401 (1970).PubMedCrossRefGoogle Scholar
  36. Osborne, N. N.: Occurrence of GABA and Taurine in the nervous system of the dogfish and some invertebrates. Comp. Pharmac. 2, 433–438 (1971a).CrossRefGoogle Scholar
  37. Osborne, N.N.: A micro-chromatographic method for the detection of biologically active monoamines of isolated neurons. Experientia (Basel) 27, 1502–1503 (1971b).CrossRefGoogle Scholar
  38. Osborne, N.N.: The in vivo synthesis of serotonin in an identified serotonin-containing neuron of Helix pomatia. Int. J. Neurosci. 3, 215–228 (1972a).CrossRefGoogle Scholar
  39. Osborne, N.N.: Effect of electrical stimulation on the in vivo metabolism of glucose and glutamic acid in an identified neuron. Brain Res. 41, 237–241 (1972b).PubMedCrossRefGoogle Scholar
  40. Osborne, N.N.: Occurrence of glycine and glutamic acid in the nervous system of two fish species and some invertebrates. Comp. Biochem. Physiol. 43B, 579–585 (1972c).Google Scholar
  41. Osborne, N.N.: The analysis of amines and amino acids in micro quantities of tissue. In: Progress in neurobiology, ed. G. A. Kerkut and J.W. Phillis. Oxford-New York-London-Paris: Pergamon Press 1973 (in press).Google Scholar
  42. Osborne, N. N., Briel, G., Neuhoff, V.: Distribution of GABA and other amino acids in different tissues of the gastropod mollusc Helix pomatia, including in vitro experiments with 14C glucose and 14C glutamic acid. Int. J. Neurosci. 1, 265–272 (1971).PubMedCrossRefGoogle Scholar
  43. Osborne, N.N., Cottrell, G.A.: Distribution of biogenic amines in the slug, Limax maximus. Z. Zellforsch. 112, 15–30 (1971).PubMedCrossRefGoogle Scholar
  44. Osborne, N.N., Cottrell, G.A.: Amine and amino acid microanalysis of two identified snail neurons with known characteristics. Experientia (Basel) 28, 656–658 (1972).CrossRefGoogle Scholar
  45. Osborne, N.N., Neuhoff, V.: Neurochemical studies on characterized neurons. Naturwissenschaften 60, 78–87 (1973).PubMedCrossRefGoogle Scholar
  46. Osborne, N.N., Szczepaniak, A.C., Neuhoff, V.: Amines and amino acids in identified neurons of Helix pomatia. Int. J. Neurosc. 5, 125–131 (1973).CrossRefGoogle Scholar
  47. Petzoldt, U., Briel, G., Gottschewski, G. H. M., Neuhoff, V.: Free amino acids in the early cleavage stages of the rabbit egg. Developmental Biology 31, 38–46 (1973).PubMedCrossRefGoogle Scholar
  48. Quentin, C.-D., Neuhoff, V.: Unpublished observations (1972).Google Scholar
  49. Roberts, E., Baxter, C.F., Harreveld, A. van, Wiersma, C.A.G., Adey, W.R., Killam, K.F. (eds.): Inhibition in the nervous system and gamma-aminobutyric acid. Oxford-New York-London- Paris: Pergamon Press 1960.Google Scholar
  50. Schroeder, W.A.: The primary structure of proteins. New York: Harper and Row Publ. 1968.Google Scholar
  51. Seiler, N.: Use of dansyl reaction in biochemical analysis. In: Methods of biochemical analysis, ed. by D. Glick, vol. 18, p. 259–337. New York-London-Sydney-Toronto: Intersci. Publ. 1970.CrossRefGoogle Scholar
  52. Seiler, N., Wiechmann, M.: Quantitative Bestimmung von Aminen und von Aminosäuren als l-Dimethylamino-naphthalin-5-sulfonsäureamid auf Dünnschichtchromatogrammen. Z. analyt. Chem. 220, 109–127 (1966).CrossRefGoogle Scholar
  53. Seiler, N., Wiechmann, M.: TCL analysis of amines as their dans-derivatives. In: Progress in thin-layer chromatography and related methods, ed. by A. Niederwieser and G. Pataki, vol. I, p. 95–144. Michigan, USA: Ann Arbor science publishers 1970.Google Scholar
  54. Spivak, V.A., Scherbukhin, V.V., Orlov, V.M., Varshavsky, La. M.: Quantitative ultramicroanalysis of amino acids in the form of their DNS-Derivatives, II. On the use of the dansylation reaction for quantitative estimation of amino acids. Analyt. Biochem. 39, 271–281 (1971).PubMedCrossRefGoogle Scholar
  55. Tang, J., Hartley, B.S.: Amino acid sequences around the disulphide bridges and methionine residues of porcine pepsin. Biochem. J. 118, 611–623 (1970).PubMedGoogle Scholar
  56. Turner, G.K.: An absolute spectrofluorometer. Science 146, 183–189 (1964).PubMedCrossRefGoogle Scholar
  57. Udenfriend, S., Stein, S., Böhlen, P., Dairmann, W., Leimgruber, W., Weigele, M.: A reagent for assay of amino acids, peptides, proteins and other primary amines in the pico-mole range. Science 178, 871–872 (1972).PubMedCrossRefGoogle Scholar
  58. Ungar, G., Desiderio, D.M., Parr, W.: Isolation, identification and synthesis of a specific-behaviour- inducing brain peptide. Nature (Lond.) New Biol. 238, 198–202 (1972).CrossRefGoogle Scholar
  59. Urban, I.-S.: Versuche zur Synthese eines fluoreszierenden Reagenz zum Nachweis von Aminosäuren. Diplomarbeit, Göttingen 1972.Google Scholar
  60. Urban, I.-S., Jastorff, B., Hettler, H., Neuhoff, V.: Synthesis of a new reagent for microanalysis of amino acids and related compounds. (In preparation.)Google Scholar
  61. Wang, K.-T., Weinstein, B.: Thin-layer chromatography on polymide layers. In: Progress in thin- layer chromatography and related methods, ed. by A. Niederwieser and G. Pataki, vol. III, p. 177–231. Michigan, USA: Ann Arbor science pub. inc. 1972.Google Scholar
  62. Weber, G.: Polarization of the fluorescence of macromolecules. 2. Fluorescent conjugates of ovalbumin and bovine serum albumin. Biochem. J. 51, 155–167 (1952).PubMedGoogle Scholar
  63. Weise, M., Eisenbach, G.M.: Quantitative determination of amino acids in the 10-14 molar range by scanning microscope photometry. Experientia (Basel) 28, 245–247 (1972).CrossRefGoogle Scholar
  64. Woods, K.R., Wang, K.T.: Separation of dansyl-amino acids by polyamide layer chromatography. Biochim. biophys. Acta (Amst.) 133, 369–370 (1967).Google Scholar
  65. Zanetta, J.P., Vincendon, G., Mandel, P., Gombos, G.: The utilisation of 2-dimethylamino- naphthalene-5-sulphonyl-chloride for quantitative determination of free amino acids and partial analysis of primary structure of proteins. J. Chromat. 51, 441–458 (1970).CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1973

Authors and Affiliations

  • Volker Neuhoff
    • 1
  1. 1.Medizin (Arbeitsgruppe Neurochemie)Max-Planck-Institut für ExperimentelleGöttingenGermany

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