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, T-1, T-2, and H as well as phosphodiesterases I and II, Kinetic measurements with RNase B and RNase T-1 showed DNP-poly(A) to be a reversible competitive inhibitor with Kr equal to 1.03 and 1.05 mu M, respectively, Data on the quenching of fluorescence of RNase T-1 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 mu M or 7.0 mu M 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 in RNase-binding affinity and capacity, By titration of the derivatized beads with RNase, the first dissociation constant (K-d) and binding capacity for the bound enzyme can be determined, The Rd was found to be 0.66 mu M for RNase B and 0.48 mu M for RNase T-1; the corresponding binding capacities were found to be 21.0 x 10(-8) and 9.6 x 10(-8) mol/g, respectively.