Determination of dissociation constants of crystalline α-chymotrypsin complexes

As a first step in investigations of the properties of crystalline enzymes, the binding of indole, N-formyl-l-phenylalanine, and N-formyl-l-p-iodophenylalanine to α-chymotrypsin crystals, and the binding of indole to tosyl-α-chymotrypsin crystals, has been studied. The methods used were spectrophotometric measurements of the concentration of indole in the supernatant, or measurements of the concentration of radioactively labeled indole in both the supernatant and the crystal. The dissociation constants of the specific binding site of the crystalline enzyme have been determined for indole and N-formyl-l-phenylalanine. It was found that indole does not bind to tosyl-α-chymotrypsin crystals and that N-formyl-p-iodophenylalanine does not bind to the substrate binding site of the crystalline enzyme.