The reagent pfluorobenzenesulfonyl chloride modifies the protein side chains of tyrosine, lysine, and histidine and the a-NH2 group. The pfluorobenzenesulfonyl (Fbs-) group, identified by the 19F nuclear magnetic resonance signal, exhibits a different 19F chemical shift for each functional group modified. The Fourier-transformed spectra of the Fbs-group displayed the expected nine-line multiplet in Fbs-amino acids and simple Fbs-peptides but not in the Fbsproteins, where the resolution was less. Lysozyme. RNase, DNase, and chymotrypsin react with this reagent and each Fbs-protein exhibits a distinctive pattern of 19F NMR signals due to the label, suggesting that the reaction of the reagent varies with the reactivity of the side chains in a protein. The three major "F signals of the unfolded Fbs-RNase in 8 M urea are due to the Fbs-label on the imidazolium, (Y-NH*, and t-NH2 groups. Based upon results from amino acid and 19F NMR analyses of the tryptic-chymotryptic peptides of Fbs-RNase, portions of the imidazolium and c-NH* resonances were assigned to the Fbs-label on His-105 and Lys-41, respectively, while the (Y-NH* resonance was entirely due to the Fbs-label on the a-NH2 of Lys-I. Because Fbs-RNase has an unchanged, near-ultraviolet circular dichroism spectrum and because it retains approximately 80% of the RNase activity. the conformation of Fbs-RNase is probably not altered from the folded conformation of the native enzyme. Upon unfolding in 8 M urea or heating at 70Β°C. Fbs-RNase gave a 19F NMR spectrum differing from that of the folded Fbs-RNase. In the presence of uridylic acid, Lys-41 was the only residue protected from modification by the reagent with a concomitant reduction of the c-NH2 resonance, and the RNase thus modified was fully active. Hence, "F NMR analysis of protein, via the reaction with p-fluorobenzenesulfonyl chloride, provided not only information about the protein conformation but also direct measurements of the modification status. (cl 1985