Fluorescent Probes for Conformational States of Proteins

The conversion of pepsinogen to pepsin has been followed by means of the fluorescent probe, Z-p-toluidinonaphthalene6-sulfonate (TNS). It was found that the change in fluorescence accurately reflects the formation of active pepsin. The data on the binding of TNS to pepsinogen and pepsin suggest that the environment surrounding the fluorophore is altered during the conversion. One of the peptides released from the zymogen enhanced TN&pepsin fluorescence and inhibited enzyme activity in the pH range 4.5 to 5.5. Fluorescence titration data showed that the observed binding phenomenon satisfied a mechanism of noncompetitive interaction between an enzyme and 2 smaller molecules, TNS and the inhibitor peptide which modified TN&pepsin fluorescence. Dissociation constants of the pepsin-peptide complex, evaluated by measuring the increase in TNS fluorescence, ranged from 3 to 5 x 10e4 M in the pH range 4.5 to 5.5. Studies of the fluorescence intensity as a function of pH for the above systems suggested that the dissociation of the peptide from pepsin is essentially complete below pH 3.6. These findings suggest that binding of the activation peptide to pepsin alters the enzyme conformation near the TNS binding site.