Fluorescence studies on the interaction of dansyl-L-arginine with trypsin and trypsinogen.

The enhancement of fluorescence intensity of the dansyl group due to the formation of trypsin- or trypsinogen-dansyl-L-arginine complex was measured. Dansyl-L-arginine (L-DA) is a product in the trypsin-catalyzed hydrolysis of dansyl-L-arginine methylester. Trypsinogen was found to have only one binding site for L-DA with the dissociation constant of 6.9 x 10(-3)M, which is identical with the Michaelis constant for the trypsin-catalyzed hydrolysis of dansyl-L-arginine amide (Goto, S. and Hess, G.P., unpublished results). This finding and the results of X-ray diffraction studies (1,2) suggest that this binding site is located in the active site of the enzyme. On the other hand, the active enzyme, trypsin, was found to have at least two binding sites for L-DA. One is located in the active site. The dissociation constant for L-DA bound to this site is 6.7 x 10(-3)M. The other site is probably located in the allosteric site of trypsin. The dissociation constant for L-DA bound to this site is 4.8 x 10(-4)M.