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) x 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.
Abel
Richele L
RL
Department of Biochemistry, University of Wisconsin at Madison, Madison, Wisconsin 53706, USA.
Haigis
Marcia C
MC
Park
Chiwook
C
Raines
Ronald T
RT
D019943:Amino Acid Substitution; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli; D019793:Fluorescein / Q000378:metabolism*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D011485:Protein Binding; D011487:Protein Conformation; D012259:Ribonuclease, Pancreatic / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D012259:Ribonuclease, Pancreatic; D019793:Fluorescein