Specific binding of thionine to the active site of trypsin.

The binding of thionine (3,6-diaminophenothiazine) to trypsin is associated with a red shift in the visible absorption spectrum of the dye. Spectrophotometric titration and equilibrium dialysis measurements are consistent with the presence of a single thionine-binding site on the trypsin molecule. At pH 7 and 4O, &is8 for the trypsin-thionine complex is 1.2 X lOUs M. The dye is displaced from trypsin by substrates, competitive inhibitors, and the chemically reactive substrate analogue 1-chloro-3-tosylamido-7-aminoZ-heptanone. Thus, the thionine-binding site overlaps with the active site region in trypsin. The dye binds very weakly to trypsinogen, with little spectral perturbation. Thionine, at low concentrations, also binds at a single site on chymotrypsin and chymotrypsinogen, showing the same spectral characteristics and a &is* value of 4.5 X low4 rd at pH 7.0 and 4’. The binding of thionine to chymotrypsin is not &ected by substrates, inhibitors, or chemically reactive substrate analogues; hence, the thionine-binding site in chymotrypsin does not appear to include any part of the active site region.