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 K1 equal to 1.03 and 1.05 microM, 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 microM 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 in RNase-binding affinity and capacity. By titration of the derivatized beads with RNase, the first dissociation constant (Kd) and binding capacity for the bound enzyme can be determined. The (Kd) was found to be 0.66 microM for RNase B and 0.48 microM for RNase T1; the corresponding binding capacities were found to be 21.0 x (10)-8 and 9.6 x (10)-8 mol/g, respectively.
Rahman
M H
MH
Department of Chemistry, State University of New York, Buffalo 14260-3000, USA.
Kang
I
I
Waterbury
R G
RG
Narang
U
U
Bright
F V
FV
Wang
J H
JH
D002846:Chromatography, Affinity; D004136:Dinitrobenzenes / Q000378:metabolism; D011061:Poly A / Q000378:metabolism; D012116:Resins, Plant; D012260:Ribonucleases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification*; D012996:Solutions; D013050:Spectrometry, Fluorescence
D004136:Dinitrobenzenes; D012116:Resins, Plant; D012996:Solutions; C100855:poly(2'-O-(2,4-dinitrophenyl))poly(adenylic acid); D011061:Poly A; D012260:Ribonucleases