The Specific Binding of Biebrich Scarlet to the Active Site of a-Chymotrypsin*

This paper reports on the presence of a strong binding site for the dye Biebrich Scarlet, (6-[Z-hydroxy-l-naphthyl]azo)3,4’-azodibenzene sulfonic acid, on cr-chymotrypsin. The 1: 1 protein-dye complex is characterized by a Kdiss of 8.8 f 0.1 X 10U5 M in 0.1 M phosphate buffer at pH 7.6 and W’. Complex formation is associated with a red shift in the visible spectrum of the dye, and a characteristic difference spectrum with a maximum at 550 rnp and a Ae, of 11,700. The dye is displaced from chymotrypsin by substrates, competitive inhibitors, and active site reagents, such as N-trans-cinnamoylimidazole and phenyhnethanesulfonylfluoride. Thus, the binding site for Biebrich Scarlet overlaps the active site region in cY-chymotrypsin. The dye does not bind strongly to chymotrypsinogen, or to trypsin, and shows no significant spectral perturbation in the presence of these proteins, under the conditions used in this study.