Fluorescence Assay for the Binding of Ribonuclease A to the Ribonuclease Inhibitor Protein

Ribonuclease A (RNase A) and the ribonuclease inhibitor protein (RI) form one of the tightest known protein–protein complexes. RNase A variants and homologues, such as G88R RNase A, that retain ribonucleolytic activity in the presence of RI are toxic to cancer cells. Herein, a new and facile assay is described for measuring the equilibrium dissociation constant ( K d ) and dissociation rate constant ( k d ) for complexes of RI and RNase A. This assay is based on the decrease in fluorescence intensity that occurs when a fluorescein-labeled RNase A binds to RI. To allow time for equilibration, the assay is most readily applied to those complexes with K d values in the nanomolar range or higher. Using this assay, the value of K d for the complex of RI with fluorescein-labeled G88R RNase A was determined to be 0.55 ± 0.03 nM. In addition, the value of K d was determined for the complex of RI with unlabeled G88R RNase A to be 0.57 ± 0.05 nM by using a competition assay with fluorescein-labeled G88R RNase A. Finally, the value of k d for the complex of RI with fluorescein-labeled G88R RNase A was determined to be (7.5 ± 0.4) × 10 −3 s −1 by monitoring the increase in fluorescence intensity upon dissociation. This assay can be used to characterize complexes of RI with a wide variety of RNase A variants and homologues, including those with cytotoxic activity.