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Specific Binding of Thionine to the Active Site of Trypsin

From the ¹ From the Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, California 90024

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 4°, K_diss for the trypsin-thionine complex is 1.2 x 10^-5 m. The dye is displaced from trypsin by substrates, competitive inhibitors, and the chemically reactive substrate analogue 1-chloro-3-tosylamido-7-amino-2-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 K_diss value of 4.5 x 10^-4 m at pH 7.0 and 4°. The binding of thionine to chymotrypsin is not affected 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.

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