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

✍ Scribed by Richele L. Abel; Marcia C. Haigis; Chiwook Park; Ronald T. Raines

Publisher: Elsevier Science
Year: 2002
Tongue: English
Weight: 222 KB
Volume: 306
Category: Article
ISSN: 0003-2697
DOI: 10.1006/abio.2002.5678

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✦ Synopsis

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.

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