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Isoelectric focusing of dansylated amino acids in immobilized pH gradients
Author(s) -
BianchiBosisio Adriana,
Righetti Pier Giorgio,
Egen Ned B.,
Bier Milan
Publication year - 1986
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150070305
Subject(s) - dansyl chloride , chemistry , amino acid , fluorescence , isoelectric point , chromatography , arginine , stereochemistry , derivatization , organic chemistry , biochemistry , high performance liquid chromatography , enzyme , physics , quantum mechanics
The 21 free amino acids commonly encountered in proteins have been transformed into “carrier ampholyte” species by reacting their primary amino groups with dansyl chloride. These derivatives can thus be focused in an immobilized pH gradient covering the pH 3.1 to 4.1 interval, except for arginine, which still retains a p I of 8.8. Due to their inherent fluorescence, the dansyl derivatives are revealed in UV light, with a sensitivity of the order of 2–4 ng/mm 2 . All nearest neighbors are separated except for the following couples: Asn‐Gln, Gly‐Thr, Val‐Ile and Cys‐Cys 2 , with a resolving power, in a Δp I scale, of the order of 0.0018 pH units. Except for a few cases (notably the aromatic amino acids) the order of p I values is well correlated with the p K values of carboxyl groups, suggesting that the latter are not altered by dansylation. From the set of p K COOH ‐p I values of the different amino acids, the p K of the tertiary amino group in the dansyl label has been calculated to be 5.11 ± 0.06. Knowing the p K of the amino‐dansyl and the p I of the excess, free dansyl label (p I = 3.34) we have derived, for its sulfonic acid group, a p K of 1.57.