Proteins containing the fluorescent dansyl (l-dimethylaminonaphthalene-5-sulfonyl) group are useful in the study of fluorescence polarization, protein conformation, and antigen-antibody reactions ( 14). After a protein is labeled by reaction with dansyl chloride, the degree of substitution is usually assessed spectrophotometrically from the optical absorption of the conjugate in the region of 340 rnp. The extinction coefficient most commonly assumed for the bound dansyl group is 4.3 X 10,: M-l cm-l, a value based on the absorption properties of several dansyl compounds in 60% ethanol solutions (5).
However, there is considerable uncertainty as to the applicability of this extinction coefficient for dansyl protein conjugates. Hartley :mtl Massey (6) used equilibrium dialysis to study conjugates of chymotrypsin and ovalbumin. In their method, the number of dansyl groups bound to the protein was determined as the difference between the added and unreacted dansyl chloride, as determined spectrophotometrical!y, in the dialyzate of the original reaction mixture. The method requires careful recovery of rather small amounts of 1-dimethylaminonaphthalene-5sulfonic acid from large volumes of dialyzate and is not suited to procedures in which the unreacted dye is eliminated by charcoal treatment or column chromatography (4). The extinction coefficients founti by Hartley and Massey (6) for the bound dansyl group were in the range 3.0 to 3.4 X lo3 M-l cm-l, but these values do not seem to have becbn widely accepted. A recent review (2) pointed out only three studies utilizing these coefficients; all other studies have employed C z-x 4.3 x 10:' M-l cm-l (5j.
Spectrophotometric analysis of dansyl conjugates to determine the degree of labeling of proteins with dansyl groups is uncertain not only because of the discrepancies in the assumed extinction coefficients but