A nitrocellulose filter binding assay for ribonucleotide reductase

Protein B1, one of the two nonidentical subunits of Escherichia coli ribonucleotide reductase, contains two classes of binding sites for nucleoside triphosphates. One class (h-sites), with a high affinity for dATP ( K D = 30 n m) regulates the substrate specificity, while the other (l-sites), with a lower affinity for dATP, regulates overall activity. These classes were defined from experiments involving equilibrium dialysis. Here we describe a sensitive alternative method to measure nucleotide binding to ribonucleotide reductases that gave the same results as equilibrium dialysis. The method involves the protein-specific binding of radioactive nucleotides to nitrocellulose filters. We believe that this method will be useful in binding studies with pure reductases from sources other than E. coli, for the characterization of mutants with changed allosteric properties, and as an assay during purification of reductases containing an h-site for dATP.