Selective Removal of Ribonucleases from Solution with Covalently Anchored Macromolecular Inhibitor

• Published in: Analytical chemistry (Washington) 1996-01, Vol.68 (1), p.134-138
• Main Authors: Rahman, M. Habibur, Kang, Insug, Waterbury, Raymond G, Narang, Upvan, Bright, Frank V, Wang, Jui H
• Format: Article
• Language: English
• Subjects: Analytical biochemistry: general aspects, technics, instrumentation; Analytical, structural and metabolic biochemistry; Biochemistry; Biological and medical sciences; Chromatography, Affinity; Dinitrobenzenes - metabolism; Fundamental and applied biological sciences. Psychology; Poly A - metabolism; Resins, Plant; Ribonucleases - antagonists & inhibitors; Ribonucleases - isolation & purification; Ribonucleic acid; RNA; Solutions; Spectrometry, Fluorescence
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac9508098

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 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 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.