Dansylation of free tyrosine or of rat brain hexokinase (ATP:D-hexose 6-phosphotransferase: EC 2.7.1. I), which contains anN-terminal tyrosine residue, yields both the didansyl and N-monodansyl derivatives if N-ethylmorpholine is used in the dansylation procedure [W. R. Gray (1972) in Methods in Enzymology (Hirs, C. H. W., and Timasheff, S. N.. eds.), Vol. 25, Academic Press, New York]. If the N-ethylmorpholine is replaced by NaHCO, buffer (pH 9.51, the didansyl derivative is formed almost exclusively. and the ambiguity resulting from the formation of two derivatives from a single N-terminal residue is thereby eliminated. Therefore, a slightly modified dansylation procedure, using NaHCO, buffer is recommended: the validity of the modified procedure was demonstrated by its successful application to six different proteins having previously known N-terminal amino acids. The didansyl and 0-monodansyl derivatives of tyrosine are remarkably photolabile as compared to the w-dansyl derivatives. Unless specific precautions against unnecessary irradiation are observed, photolytic degradation of the didansyl tyrosine derivatives could occur during experimental manipulations; loss of the didansylated compound and formation of photolysis products complicates the interpretation of experiments in which a single didansyl derivative (of the N-terminal residue) is expected.
' Abbreviations used: Dansyl chloride, 5-dimethylamino-I-naphthalene sulfonyl chloride; SDS. sodium dodecyl sulfate; PPO, 2,5diphenyloxazole:
POPOP, 1,4-bis[2(5phenyloxazoyljlbenzene.
Dansylation will be used to include both the reaction with dansyl chloride, or, in reference to the determination of N-terminal amino acid residues, in the more inclusive sense of reaction with dansyl chloride followed by hydrolysis and subsequent identification of dansyl derivatives.
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