Poly[2'-
O-(2,4-dinitrophenyl)]poly(A)[DNP-poly(A)] hasbeen found to be a potent inhibitor in solution for RNasesA, B, S, T
1, T
2, and H as well asphosphodiesterases Iand II. Kinetic measurements with RNase B and RNaseT
1 showed DNP-poly(A) to be a reversiblecompetitiveinhibitor with
KI equal to 1.03 and 1.05
M,respectively.Data on the quenching of fluorescence of RNase T
1byDNP-poly(A) indicate the existence of more than oneRNase-binding site in each DNP-poly(A) molecule.Byattaching each DNP-poly(A) molecule at one end covalently to oxirane acrylic beads, an affinity column wasprepared for selective removal of RNases from aqueoussolutions by simple filtration. It was found that a1000-fold reduction in RNase concentration can be obtainedby passing either 7.0
M or 7.0 nM RNase A solutionthrough a 5-cm-long column. The column can be saturated by passing through a concentrated RNase solutionand subsequently regenerated by washing with salt solution. The regenerated column can be used repeatedlywith no significant decrease in RNase-binding affinity andcapacity. By titration of the derivatized beads withRNase,the first dissociation constant (
Kd) and bindingcapacityfor the bound enzyme can be determined. The
Kd wasfound to be 0.66
M for RNase B and 0.48
M for RNaseT
1; the corresponding binding capacities were foundtobe 21.0 × 10
-8 and 9.6 ×10
-8 mol/g, respectively.