Specific fluorescent derivatives of macromolecules. Reaction of dansyl fluoride with serine proteinases.
5-Dimethylaminonaphthalene-1-sulfonyl fluoride was evaluated as a reagent for the selective labeling of proteins. In a comparative study with Dns-chloride a greatly increased selectivity of the fluoride was found with a number of proteins. The reaction of Dns-fluoride with alpha-chymotrypsin, subtilisin Carlsberg and trypsin was found to be highly specific, resulting in a stoichiometric incorporation of the Dns label with concomitant loss of enzymatic activity. The reaction of Dns-chloride with the same proteinases is unspecific. Evidence was obtained to indicate that reaction of the serine esterases with Dns-fluoride occurs exclusively at the active serine residue. The stability of Dns-fluoride labeled chymotrypsin was investigated. The conjugate was found to be fairly stable in the pH range from 3 to 9 at 25 degrees C and is therefore suitable for fluorescence investigations of the chymotrypsin active-site. Molar extinction coefficients for Dns-labeled serine proteinases were determined using radiocative label.
Specific fluorescent derivatives of macromolecules. Reaction of dansyl fluoride with serine proteinases. 5-Dimethylaminonaphthalene-1-sulfonyl fluoride was evaluated as a reagent for the selective labeling of proteins. In a comparative study with Dns-chloride a greatly increased selectivity of the fluoride was found with a number of proteins. The reaction of Dns-fluoride with alpha-chymotrypsin, subtilisin Carlsberg and trypsin was found to be highly specific, resulting in a stoichiometric incorporation of the Dns label with concomitant loss of enzymatic activity. The reaction of Dns-chloride with the same proteinases is unspecific. Evidence was obtained to indicate that reaction of the serine esterases with Dns-fluoride occurs exclusively at the active serine residue. The stability of Dns-fluoride labeled chymotrypsin was investigated. The conjugate was found to be fairly stable in the pH range from 3 to 9 at 25 degrees C and is therefore suitable for fluorescence investigations of the chymotrypsin active-site. Molar extinction coefficients for Dns-labeled serine proteinases were determined using radiocative label.