Isoelectric focusing of dansylated amino acids in immobilized pH gradients

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.