Selective removal of ribonucleases from solution with covalently anchored macromolecular inhibitor

Poly[2′-O-(2,4-dinitrophenyl)]poly(A) [DNP-poly(A)] has been found to be a potent inhibitor in solution for RNases A, B, S, T₁, T₂, and H as well as phosphodiesterases I and II. Kinetic measurements with RNase B and RNase T₁ showed DNP-poly(A) to be a reversible competitive inhibitor with K_I equal to 1.03 and 1.05 μM, respectively. Data on the quenching of fluorescence of RNase T₁ by DNP-poly(A) indicate the existence of more than one RNase-binding site in each DNP-poly(A) molecule. By attaching each DNP-poly(A) molecule at one end covalently to oxirane acrylic beads, an affinity column was prepared for selective removal of RNases from aqueous solutions by simple filtration. It was found that a 1000-fold reduction in RNase concentration can be obtained by passing either 7.0 μM or 7.0 nM RNase A solution through a 5-cm-long column. The column can be saturated by passing through a concentrated RNase solution and subsequently regenerated by washing with salt solution. The regenerated column can be used repeatedly with no significant decrease hi RNase-binding affinity and capacity. By titration of the derivatized beads with RNase, the first dissociation constant (Ed) and binding capacity for the bound enzyme can be determined. The K_d was found to be 0.66 μM for RNase B and 0.48 μM for RNase T₁; the corresponding binding capacities were found to be 21.0 × 10^-8 and 9.6 × 10^-8 mol/g, respectively.